AU2005242196A1 - Expression cassettes for meristem-preferential expression in plants - Google Patents

Description

P001 Section 29 Regulation 3.2(2)

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: Expression cassettes for meristem-preferential expression in plants The following statement is a full description of this invention, including the best method of performing it known to us: PF 56134 i' Expression cassettes for meristem-preferential expression in plants

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FIELD OF THE INVENTION SThe present invention relates to expression cassettes comprising transcription regulat- O ing nucleotide sequences with meristem-preferential or meristem-specific expression profiles in plants obtainable from Arabidopsis thaliana genes At2g02180, At5g54510, 0 At2g26970, At2g01180, At3g45560, At4g00580, At1g54480, or At4g11490, or the SArabidopsis thaliana genomic sequences as described by SEQ ID NO: 35 or 36.

\BACKGROUND OF THE INVENTION Manipulation of plants to alter and/or improve phenotypic characteristics (such as pro- Cl ductivity or quality) requires the expression of heterologous genes in plant tissues.

Such genetic manipulation relies on the availability of a means to drive and to control n gene expression as required. For example, genetic manipulation relies on the availabil- O ity and use of suitable promoters which are effective in plants and which regulate gene S 15 expression so as to give the desired effect(s) in the transgenic plant.

The plant meristem is the source from which new tissues and organs of a plant are produced. The meristem-preferential or meristem-specific promoters are useful for regulating plant development and other relevant agronomic traits. However, the number of promoters with meristem-preferential or meristem-specific expression profiles is very limited.

There is, therefore, a great need in the art for the identification of novel sequences that can be used for expression of selected transgenes in economically important plants. It is thus an objective of the present invention to provide new and alternative expression cassettes for meristem-preferential or meristem-specific expression of transgenes in plants. The objective is solved by the present invention.

SUMMARY OF THE INVENTION Accordingly, a first embodiment of the invention relates to an expression cassette for meristem-specific or meristem-preferential transcription of an operatively linked nucleic acid sequence in plants comprising i) at least one transcription regulating nucleotide sequence of a plant gene, said plant gene selected from the group of genes described by the GenBank Arabidopsis thaliana genome loci At2g02180, At5g54510, At2g26970, At2g01180, At3g45560, At4g00580, At1g54480, or At4g11490, or the Arabidopsis thaliana genomic sequences as described by SEQ ID NO: 35 or 36, or a functional equivalent thereof, and functionally linked thereto ii) at least one nucleic acid sequence which is heterologous in relation to said transcription regulating nucleotide sequence.

PF 56134 2 SPreferably, the transcription regulating nucleotide sequence (or the functional equiva- O lent thereof) is selected from the group of sequences consisting of CN i) the sequences described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, O 21, 24, 25, 28, 31, 32, 35, and 36, ii) a fragment of at least 50 consecutive bases of a sequence under i) which has substantially the same promoter activity as the corresponding transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 21, 24, 25, 28, 31, 32, 35, or 36; I iii) a nucleotide sequence having substantial similarity with a sequence identity of at least 40, 50, 60, to 70%, preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, 81% to 84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and C-l 99%) to a transcription regulating nucleotide sequence described by SEQ ID NO: 1,2,3,6,7,8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, or 36; iv) a nucleotide sequence capable of hybridizing (preferably under conditions equiva- Lc- lent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 2 X SSC, 0. 1% SDS at 50 0 C (more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 1 X SSC, 0.1% SDS at 50*C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 0.5 X SSC, 0. 1% SDS at 50 0 C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 0.1 X SSC, 0.1% SDS at 50*C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50"C with washing in 0.1 X SSC, 0.1% SDS at 65 0 C) to a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 28, 31, 32, 35, or 36, or the complement thereof; v) a nucleotide sequence capable of hybridizing (preferably under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 2 X SSC, 0. 1% SDS at 50 0 C (more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 1 X SSC, 0.1% SDS at 50 0 C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 0.5 X SSC, 0. 1% SDS at 50 0 C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 0.1 X SSC, 0.1% SDS at 50 0 C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 0.1 X SSC, 0.1% SDS at 65 0 C) to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, and 36, or the complement thereof; vi) a nucleotide sequence which is the complement or reverse complement of any of the previously mentioned nucleotide sequences under i) to v).

The functional equivalent of the transcription regulating nucleotide sequence is obtained or obtainable from plant genomic DNA from a gene encoding a polypeptide which has at least 70% amino acid sequence identity to a polypeptide selected from the group described by SEQ ID NO: 5, 10, 14, 19, 23, 27, 30, and 34, respectively.

PF 56134 3 Vn The expression cassette may be employed for numerous expression purposes such as for example expression of a protein, or expression of a antisense RNA, sense or dou- (cN ble-stranded RNA. Preferably, expression of the nucleic acid sequence confers to the Splant an agronomically valuable trait.

Other embodiments of the invention relate to vectors comprising an expression cassette of the invention, and transgenic host cell or non-human organism comprising an expression cassette or a vector of the invention. Preferably the organism is a plant.

IN 10 Another embodiment of the invention relates to a method for identifying and/or isolating Sa sequence with meristem-specific or meristem-preferential transcription regulating N, activity characterized that said identification and/or isolation utilizes a nucleic acid sequence encoding a amino acid sequence as described by SEQ ID NO: 5, 10, 14, 19, I23, 27, 30, or 34 or a part of at least 15 bases thereof. Preferably the nucleic acid se- 0 15 quences is described by SEQ ID NO: 4, 9, 13, 18, 22, 26, 29, or 33 or a part of at least bases thereof. More preferably, identification and/or isolation is realized by a method selected from polymerase chain reaction, hybridization, and database screening.

Another embodiment of the invention relates to a method for providing a transgenic expression cassette for meristem-specific or meristem-preferential expression comprising the steps of: I. isolating of a meristem-preferential or meristem-specific transcription regulating nucleotide sequence utilizing at least one nucleic acid sequence or a part thereof, wherein said sequence is encoding a polypeptide described by SEQ ID NO: 5, 14, 19, 23, 27, 30, or 34, or a part of at least 15 bases thereof, and II. functionally linking said meristem-preferential or meristem-specific transcription regulating nucleotide sequence to another nucleotide sequence of interest, which is heterologous in relation to said meristem-preferential or meristem-specific transcription regulating nucleotide sequence.

DEFINITIONS

It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, plant species or genera, constructs, and reagents described as such. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for exampie, reference to "a vector" is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art, and so forth.

The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 per-cent up or down (higher or lower).

PF 56134 4 n As used herein, the word "or" means any one member of a particular list and also in- 8 cludes any combination of members of that list.

SThe term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. For example, gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences. Genes also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be O 10 obtained from a variety of sources, including cloning from a source of interest or syn- _thesizing from known or predicted sequence information, and may include sequences N' designed to have desired parameters.

SThe term "native" or "wild type" gene refers to a gene that is present in the genome of an untransformed cell, a cell not having a known mutation.

A "marker gene" encodes a selectable or screenable trait.

The term "chimeric gene" refers to any gene that contains 1) DNA sequences, including regulatory and coding sequences, that are not found together in nature, or 2) sequences encoding parts of proteins not naturally adjoined, or 3) parts of promoters that are not naturally adjoined.

Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences.

and coding sequences derived from the same source, but arranged in a manner different from that found in nature.

A "transgene" refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed.

Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.

An "oligonucleotide" corresponding to a nucleotide sequence of the invention, for use in probing or amplification reactions, may be about 30 or fewer nucleotides in length 9, 12, 15, 18, 20, 21 or 24, or any number between 9 and 30). Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16 to 24 nucleotides in length may be preferred. Those skilled in the art are well versed in the design of primers for use processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 100's or even 1000's of nucleotides in length.

The terms "protein," "peptide" and "polypeptide" are used interchangeably herein.

PF 56134 V)The nucleotide sequences of the invention can be introduced into any plant. The genes o to be introduced can be conveniently used in expression cassettes for introduction and N expression in any plant of interest. Such expression cassettes will comprise the tran- O scriptional initiation region of the invention linked to a nucleotide sequence of interest.

Preferred promoters include constitutive, tissue-specific, developmental-specific, inducible and/or viral promoters, most preferred are the meristem-specific or meristempreferential promoters of the invention. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally O 10 contain selectable marker genes. The cassette will include in the direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interr est, and a transcriptional and translational termination region functional in plants. The termination region may be native with the transcriptional initiation region, may be native Swith the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such, as the octopine synthase and nopaline synthase termination regions (see also, Guerineau 1991; Proudfoot 1991; Sanfacon 1991; Mogen 1990; Munroe 1990; Ballas 1989; Joshi 1987).

"Coding sequence" refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an "uninterrupted coding sequence", lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions. An "intron" is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.

The terms "open reading frame" and "ORF" refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. The terms "initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides ('codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).

A "functional RNA" refers to an antisense RNA, ribozyme, or other RNA that is not translated.

The term "RNA transcript" refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. "Messenger RNA" (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA.

,,Transcription regulating nucleotide sequence", transcription regulating nucleotide sequence "regulatory sequences", and "suitable regulatory sequences", each refer to PF 56134 6 Snucleotide sequences influencing the transcription, RNA processing or stability, or 8 translation of the associated (or functionally linked) nucleotide sequence to be trancscribed. The transcription regulating nucleotide sequence may have various localiza- Stions with the respect to the nucleotide sequences to be transcribed. The transcription regulating nucleotide sequence may be located upstream non-coding sequences), within, or downstream non-coding sequences) of the sequence to be transcribed a coding sequence). The transcription regulating nucleotide sequences may be selected from the group comprising enhancers, promoters, translation leader sequences, introns, 5'-untranslated sequences, 3'-untranslated sequences, and polyade- IO 10 nylation signal sequences. They include natural and synthetic sequences as well as sequences, which may be a combination of synthetic and natural sequences. As is Lc' noted above, the term "transcription regulating nucleotide sequence" is not limited to promoters. However, preferably a transcription regulating nucleotide sequence of the Iinvention comprises at least one promoter sequence a sequence localized up- 0 15 stream of the transcription start of a gene capable to induce transcription of the downstream sequences). In one preferred embodiment the transcription regulating nucleotide sequence of the invention comprises the promoter sequence of the corresponding gene and optionally and preferably the native 5'-untranslated region of said gene.

Furthermore, the 3'-untranslated region and/or the polyadenylation region of said gene may also be employed.

non-coding sequence" refers to a nucleotide sequence located 5' (upstream) to the coding sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency (Turner 1995).

non-coding sequence" refers to nucleotide sequences located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.

The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al., 1989.

The term "translation leader sequence" refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream of the translation start codon. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.

"Signal peptide" refers to the amino terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into the secretory pathway. The term "signal sequence" refers to a nucleotide sequence that encodes the signal peptide. The term "transit peptide" as used herein refers part of a expressed polypeptide (preferably to the amino terminal extension of a polypeptide), which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into a cell organelle

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PF 56134 7 S(such as the plastids chloroplasts) or mitochondria). The term "transit sequence" O refers to a nucleotide sequence that encodes the transit peptide.

S"Promoter" refers to a nucleotide sequence, usually upstream to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA.

This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" Sis a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements, derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.

The "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters." In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A "minimal or core promoter" thus consists only of all basal elements needed for transcription initiation, a TATA box and/or an initiator.

"Constitutive expression" refers to expression using a constitutive or regulated promoter. "Conditional" and "regulated expression" refer to expression controlled by a regulated promoter.

"Constitutive promoter" refers to a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant. Each of the transcription-activating elements do not exhibit an absolute tissue-specificity, but mediate transcriptional activation in most plant PF 56134 8 parts at a level of at least 1% of the level reached in the part of the plant in which transcription is most active.

0 "Regulated promoter" refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and includes both tissue-specific and inducible promoters. It includes natural and synthetic sequences as well as se- Squences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.

ID 10 New promoters of various types useful in plant cells are constantly being discovered, _numerous examples may be found in the compilation by Okamuro et al. (1989). Typical N regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters de- Srived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid-inducible system, promoters derived from Spathogen-inducible systems, and promoters derived from ecdysone-inducible systems.

"Tissue-specific promoter" refers to regulated promoters that are not expressed in all plant cells but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells). These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence.

"Inducible promoter" refers to those regulated promoters that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, or a pathogen.

"Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.

"Expression" refers to the transcription and/or translation of an endogenous gene, ORF or portion thereof, or a transgene in plants. For example, in the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. In addition, expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of protein.

"Specific expression" is the expression of gene products which is limited to one or a few plant tissues (spatial limitation) and/or to one or a few plant developmental stages (temporal limitation). It is acknowledged that hardly a true specificity exists: promoters seem to be preferably switch on in some tissues, while in other tissues there can be no r PF 56134 9 Sor only little activity. This phenomenon is known as leaky expression. However, with 8 specific expression in this invention is meant preferable expression in one or a few Splant tissues.

S 5 The "expression pattern" of a promoter (with or without enhancer) is the pattern of expression levels which shows where in, the plant and in what developmental stage transcription is initiated by said promoter. Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter. The level of expression of a O 10 promoter can be determined by measuring the 'steady state' concentration of a standard transcribed reporter mRNA. This measurement is indirect since the concentration N of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded. Therefore, the steady state level is the product of Ssynthesis rates and degradation rates.

The rate of degradation can however be considered to proceed at a fixed rate when the transcribed sequences are identical, and thus this value can serve as a measure of synthesis rates. When promoters are compared in this way techniques available to those skilled in the art are hybridization SI-RNAse analysis, northern blots and competitive RT-PCR. This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA.

The analysis of transcription start points in practically all promoters has revealed that there is usually no single base at which transcription starts, but rather a more or less clustered set of initiation sites, each of which accounts for some start points of the mRNA. Since this distribution varies from promoter to promoter the sequences of the reporter mRNA in each of the populations would differ from each other. Since each mRNA species is more or less prone to degradation, no single degradation rate can be expected for different reporter mRNAs. It has been shown for various eukaryotic promoter sequences that the sequence surrounding the initiation site ('initiator') plays an important role in determining the level of RNA expression directed by that specific promoter. This includes also part of the transcribed sequences. The direct fusion of promoter to reporter sequences would therefore lead to suboptimal levels of transcription.

A commonly used procedure to analyze expression patterns and levels is through determination of the 'steady state' level of protein accumulation in a cell. Commonly used candidates for the reporter gene, known to those skilled in the art are betaglucuronidase (GUS), chloramphenicol acetyl transferase (CAT) and proteins with fluorescent properties, such as green fluorescent protein (GFP) from Aequora victoria. In principle, however, many more proteins are suitable for this purpose, provided the protein does not interfere with essential plant functions. For quantification and determination of localization a number of tools are suited. Detection systems can readily be created or are available which are based on, immunochemical, enzymatic, fluorescent detection and quantification. Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression.

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PF 56134 n Generally, individual transformed lines with one chimeric promoter reporter construct 8 will vary in their levels of expression of the reporter gene. Also frequently observed is N the phenomenon that such transformants do not express any detectable product (RNA or protein). The variability in expression is commonly ascribed to 'position effects', although the molecular mechanisms underlying this inactivity are usually not clear.

"Overexpression" refers to the level of expression in transgenic cells or organisms that exceeds levels of expression in normal or untransformed (non-transgenic) cells or organisms.

ID "Antisense inhibition" refers to the production of antisense RNA transcripts capable of N suppressing the expression of protein from an endogenous gene or a transgene.

S"Gene silencing" refers to homology-dependent suppression of viral genes, transgenes, or endogenous nuclear genes. Gene silencing may be transcriptional, when the suppression is due to decreased transcription of the affected genes, or post-transcriptional, when the suppression is due to increased turnover (degradation) of RNA species homologous to the affected genes (English 1996). Gene silencing includes virus-induced gene silencing (Ruiz et al. 1998).

The terms "heterologous DNA sequence," "exogenous DNA segment" or "heterologous nucleic acid," as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

"Homologous to" in the context of nucleotide sequence identity refers to the similarity between the nucleotide sequence of two nucleic acid molecules or between the amino acid sequences of two protein molecules. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (as described in Haines and Higgins Nucleic Acid Hybridization, IRL Press, Oxford, or by the comparison of sequence similarity between two nucleic acids or proteins.

The term "substantially similar" refers to nucleotide and amino acid sequences that represent functional and/or structural equivalents of Arabidopsis sequences disclosed herein.

In its broadest sense, the term "substantially similar" when used herein with respect to a nucleotide sequence means that the nucleotide sequence is part of a gene which encodes a polypeptide having substantially the same structure and function as a poly- PF 56134 11 peptide encoded by a gene for the reference nucleotide sequence, the nucleotide 8 sequence comprises a promoter from a gene that is the ortholog of the gene corre- (N sponding to the reference nucleotide sequence, as well as promoter sequences that Sare structurally related the promoter sequences particularly exemplified herein, the substantially similar promoter sequences hybridize to the complement of the promoter sequences exemplified herein under high or very high stringency conditions. For example, altered nucleotide sequences which simply reflect the degeneracy of the genetic code but nonetheless encode amino acid sequences that are identical to a particular amino acid sequence are substantially similar to the particular sequences. The ID 10 term "substantially similar" also includes nucleotide sequences wherein the sequence _has been modified, for example, to optimize expression in particular cells, as well as nucleotide sequences encoding a variant polypeptide having one or more amino acid substitutions relative to the (unmodified) polypeptide encoded by the reference sequence, which substitution(s) does not alter the activity of the variant polypeptide relative to the unmodified polypeptide.

In its broadest sense, the term "substantially similar" when used herein with respect to polypeptide means that the polypeptide has substantially the same structure and function as the reference polypeptide. In addition, amino acid sequences that are substantially similar to a particular sequence are those wherein overall amino acid identity is at least 65% or greater to the instant sequences. Modifications that result in equivalent nucleotide or amino acid sequences are well within the routine skill in the art. The percentage of amino acid sequence identity between the substantially similar and the reference polypeptide is at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to at least 99%, wherein the reference polypeptide is an Arabidopsis polypeptide encoded by a gene with a promoter having any one of SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, or 36, a nucleotide sequence comprising an open reading frame having any one of SEQ ID NOs: 4, 9, 13, 18, 22, 26, 29, or 33, which encodes one of SEQ ID NOs: 5, 10, 14, 19, 23, 27, 30, or 34. One indication that two polypeptides are substantially similar to each other, besides having substantially the same function, is that an agent, an antibody, which specifically binds to one of the polypeptides, also specifically binds to the other.

Sequence comparisons maybe carried out using a Smith-Waterman sequence alignment algorithm (see Waterman (1995)). The localS program, version 1.16, is preferably used with following parameters: match: 1, mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2.

Moreover, a nucleotide sequence that is "substantially similar" to a reference nucleotide sequence is said to be "equivalent" to the reference nucleotide sequence. The skilled artisan recognizes that equivalent nucleotide sequences encompassed by this invention can also be defined by their ability to hybridize, under low, moderate and/or stringent conditions 0.1 X SSC, 0.1% SDS, 65°C), with the nucleotide sequences that are within the literal scope of the instant claims.

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PF 56134 12 SWhat is meant by "substantially the same activity" when used in reference to a polynucleotide or polypeptide fragment is that the fragment has at least 65%, 66%, 67%, C- 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, S82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, 91%, 92%, 5 93%, 94%, 95%, 96%, 97%, 98%, up to at least 99% of the activity of the full length polynucleotide or full length polypeptide.

"Target gene" refers to a gene on the replicon that expresses the desired target coding sequence, functional RNA, or protein. The target gene is not essential for replicon rep- O 10 lication. Additionally, target genes may comprise native non-viral genes inserted into a non-native organism, or chimeric genes, and will be under the control of suitable regu- C-i latory sequences. Thus, the regulatory sequences in the target gene may come from any source, including the virus. Target genes may include coding sequences that are Seither heterologous or homologous to the genes of a particular plant to be transformed.

However, target genes do not include native viral genes. Typical target genes include, but are not limited to genes encoding a structural protein, a seed storage protein, a protein that conveys herbicide resistance, and a protein that conveys insect resistance.

Proteins encoded by target genes are known as "foreign proteins". The expression of a target gene in a plant will typically produce an altered plant trait.

The term "altered plant trait" means any phenotypic or genotypic change in a transgenic plant relative to the wild-type or non-transgenic plant host.

"Replication gene" refers to a gene encoding a viral replication protein. In addition to the ORF of the replication protein, the replication gene may also contain other overlapping or non-overlapping ORF(s), as are found in viral sequences in nature. While not essential for replication, these additional ORFs may enhance replication and/or viral DNA accumulation. Examples of such additional ORFs are AC3 and AL3 in ACMV and TGMV geminiviruses, respectively.

"Chimeric trans-acting replication gene" refers either to a replication gene in which the coding sequence of a replication protein is under the control of a regulated plant promoter other than that in the native viral replication gene, or a modified native viral replication gene, for example, in which a site specific sequence(s) is inserted in the 5' transcribed but untranslated region. Such chimeric genes also include insertion of the known sites of replication protein binding between the promoter and the transcription start site that attenuate transcription of viral replication protein gene.

"Chromosomally-integrated" refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes are not "chromosomally integrated" they may be "transiently expressed." Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus.

The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the r PF 56134 13 V transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms". Examples of CN methods of transformation of plants and plant cells include Agrobacterium-mediated O transformation (De Blaere 1987) and particle bombardment technology (US 4,945,050).

Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan (see, for example, Fromm 1990).

"Transformed," "transgenic," and "recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.

0 10 The nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis Gelfand 1999. Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific Iprimers, vector-specific primers, partially mismatched primers, and the like. For exam- S 15 pie, "transformed," "transformant," and "transgenic" plants or calli have been through the transformation process and contain a foreign gene integrated into their chromosome. The term "untransformed" refers to normal plants that have not been through the transformation process.

"Transiently transformed" refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium-mediated transformation or biolistic bombardment), but not selected for stable maintenance.

"Stably transformed" refers to cells that have been selected and regenerated on a selection media following transformation.

"Transient expression" refers to expression in cells in which a virus or a transgene is introduced by viral infection or by such methods as Agrobacterium-mediated transformation, electroporation, or biolistic bombardment, but not selected for its stable maintenance.

"Genetically stable" and "heritable" refer to chromosomally-integrated genetic elements that are stably maintained in the plant and stably inherited by progeny through successive generations.

"Primary transformant" and 'TO generation" refer to transgenic plants that are of the same genetic generation as the tissue which was initially transformed not having gone through meiosis and fertilization since transformation).

"Secondary transformants" and the "T1, T2, T3, etc. generations" refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants.

'Wild-type" refers to a virus or organism found in nature without any known mutation.

PF 56134 14 S"Genome" refers to the complete genetic material of an organism.

N, The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers Sthereof in either single- or double-stranded form, composed of monomers (nucleotides) S 5 containing a sugar, phosphate and a base which is either a purine or pyrimidine.

Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encom- O 10 passes conservatively modified variants thereof degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with Smixed-base and/or deoxyinosine residues (Batzer 1991; Ohtsuka 1985; Rossolini 1994). A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. In higher plants, deoxyribonucleic acid (DNA) is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms "nucleic acid" or "nucleic acid sequence" may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.

The invention encompasses isolated or substantially purified nucleic acid or protein compositions. In the context of the present invention, an "isolated" or "purified" DNA molecule or an "isolated" or "purified" polypeptide is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.

For example, an "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein of interest chemicals.

PF 56134 SThe nucleotide sequences of the invention include both the naturally occurring se- 8 quences as well as mutant (variant) forms. Such variants will continue to possess the desired activity, either promoter activity or the activity of the product encoded by Sthe open reading frame of the non-variant nucleotide sequence.

The term "variant" with respect to a sequence a polypeptide or nucleic acid sequence such as for example a transcription regulating nucleotide sequence of the invention) is intended to mean substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences that, ID 10 because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be Sidentified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein. Generally, nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, 81%- 84%, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, to 98% and 99% nucleotide sequence identity to the native (wild type or endogenous) nucleotide sequence.

"Conservatively modified variations" of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations" which are one species of "conservatively modified variations." Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

The nucleic acid molecules of the invention can be "optimized" for enhanced expression in plants of interest (see, for example, WO 91/16432; Perlak 1991; Murray 1989).

In this manner, the open reading frames in genes or gene fragments can be synthesized utilizing plant-preferred codons (see, for example, Campbell Gowri, 1990 for a discussion of host-preferred codon usage). Thus, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized se- PF 56134 16 t quences may also be used. Variant nucleotide sequences and proteins also encom- 8 pass, sequences and protein derived from a mutagenic and recombinogenic procedure 0c such as DNA shuffling. With such a procedure, one or more different coding sequences 0 can be manipulated to create a new polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. Strategies for such DNA shuffling are known in the art (see, for example, Stemmer 1994; Stemmer 1994; Crameri 1997; Moore 1997; Zhang 1997; Crameri 1998; and US 5,605,793 and 5,837,458).

C, By "variant" polypeptide is intended a polypeptide derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal t and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

Thus, the polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art (see, for example, Kunkel 1985; Kunkel 1987; US 4,873,192; Walker Gaastra, 1983 and the references cited therein). Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978). Conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred.

Individual substitutions deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than more typically less than in an encoded sequence are "conservatively modified variations," where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: Aliphatic: Glycine Alanine Valine Leucine Isoleucine Aromatic: Phenylalanine Tyrosine Tryptophan Sulfur-containing: Methionine Cysteine Basic: Arginine Lysine Histidine Acidic: Aspartic acid Glutamic acid Asparagine Glutamine See also, Creighton, 1984. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservatively modified variations." "Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to a nucleotide sequence of interest, which is optionally PF 56134 17 Soperably linked to termination signals and/or other regulatory elements. An expression cassette may also comprise sequences required for proper translation of the nucleotide C sequence. The coding region usually codes for a protein of interest but may also code U for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in C 5 the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one,'which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. An expression cassette may be as- O 10 sembled entirely extracellularly by recombinant cloning techniques). However, an expression cassette may also be assembled using in part endogenous components.

C For example, an expression cassette may be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence, which thereby becomes func- Stionally linked and controlled by said promoter sequences. Likewise, a nucleic acid sequence to be expressed may be placed (or inserted) downstream of an endogenous Spromoter sequence thereby forming an expression cassette. The expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development the meristem-specific or meristem-preferential promoters of the invention).

"Vector" is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g.

autonomous replicating plasmid with an origin of replication).

Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic higher plant, mammalian, yeast or fungal cells).

Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.

"Cloning vectors" typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with

I

PF 56134 18 Sthe cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance.

A "transgenic plant" is a plant having one or more plant cells that contain an expression S 5 vector.

"Plant tissue" includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture such as single cells, protoplast, embryos, and callus tissue.

O 10 The plant tissue may be in plants or in organ, tissue or cell culture.

C The following terms are used to describe the sequence relationships between two or c- more nucleic acids or polynucleotides: "reference sequence", "comparison win- Sdow", "sequence identity", "percentage of sequence identity", and "substantial identity".

As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA or gene sequence, or the complete cDNA or gene sequence.

As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art.

Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Preferred, non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, 1988; the local homology algorithm of Smith et al. 1981; the homology alignment algorithm of Needleman and Wunsch 1970; the search-for-similarity-method of Pearson and Lipman 1988; the algorithm of Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be per- PF 56134 19 n formed using the default parameters. The CLUSTAL program is well described 8 (Higgins 1988, 1989; Corpet 1988; Huang 1992; Pearson 1994). The ALIGN pro- Sgram is based on the algorithm of Myers and Miller, supra. The BLAST programs of Altschul et al., 1990, are based on the algorithm of Karlin and Altschul, supra.

S Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Alt- Sschul 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in Sboth directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always and N (penalty score for mismatching residues; always For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, Karlin Altschul (1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST can be utilized as described in Altschul et al. 1997. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al., supra. When utilizing BLAST; Gapped BLAST, PSI-BLAST, the default parameters of the respective programs BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength of 11, an expectation of 10, a cutoff of 100, M=5, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (WV) of 3, an expectation of 10, and the BLOSUM62 scoring matrix (see Henikoff Henikoff, 1989). See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.

I

PF 56134 SFor purposes of the present invention, comparison of nucleotide sequences for de- 8 termination of percent sequence identity to the promoter sequences disclosed Sherein is preferably made using the BlastN program (version 1.4.7 or later) with its Sdefault parameters or any equivalent program. By "equivalent program" is intended S 5 any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program.

ID 10 As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences Sthat are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to Sproteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).

As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

The term "substantial identity" or "substantial similarity" of polynucleotide sequences (preferably for a protein encoding sequence) means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described usr PF 56134 21 Sing standard parameters. The term "substantial identity" or "substantial similarity" 8 of polynucleotide sequences (preferably for promoter sequence) means (as described above for variants) that a polynucleotide comprises a sequence that has at Sleast 40, 50, 60, to 70%, preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, 81%-84%, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corre- ID 10 sponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, more preferably at least 80%, 90%, and Smost preferably at least Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions (see below). Generally, stringent conditions are selected to be about 5 0 C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 10C to about 200C, depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

(ii) The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and VWunsch (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.

The sequence comparison algorithm then calculates the percent sequence identity for PF 56134 22 Sthe test sequence(s) relative to the reference sequence, based on the designated proo gram parameters.

SAs noted above, another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture total cellular) DNA or RNA.

"Bind(s) substantially" refers to complementary hybridization between a probe nucleic O 10 acid and a target nucleic acid and embraces minor mismatches that can be accommo- _dated by reducing the stringency of the hybridization media to achieve the desired de- Stection of the target nucleic acid sequence.

I"Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence dependent, and are different under different environmental parameters. The Tm is the temperature (under defined ionic strength and pH) at which of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, 1984: Tm 81.5°C 16.6 (logio M)+0.41 0.61 form)- 500 L where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is reduced by about I 1C for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10 0 C. Generally, stringent conditions are selected to be about 5 0 C lower than the thermal melting point I for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4 0 C lower than the thermal melting point I; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point I; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, or 20 0 C lower than the thermal melting point I. Using the equation, hybridization and wash compositions, and desired T, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T of less than 45 0 C (aqueous solution) or 32 0 C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, 1993. Generally, highly stringent hybridization and wash conditions are selected to be about 5 0 C lower than the thermal melting point Tm for the specific sequence at a defined ionic strength and pH.

r PF 56134 23 SAn example of highly stringent wash conditions is 0.15 M NaCI at 72 0 C for about Sminutes. An example of stringent wash conditions is a 0.2 X SSC wash at 65°C for C1 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high strin- O gency wash is preceded by a low stringency wash to remove background probe signal.

An example medium stringency wash for a duplex of, more than 100 nucleotides, is 1 X SSC at 45*C for 15 minutes. An example low stringency wash for a duplex of, more than 100 nucleotides, is 4 to 6 X SSC at 40°C for 15 minutes. For short probes about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion con- I 10 centration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30 0 C and at least about 60 0 C for long robes >50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2 X (or higher) than that observed for an Iunrelated probe in the particular hybridization assay indicates detection of a specific S 15 hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37 0

C,

and a wash in 0.1 x SSC at 60 to 65°C. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1% SDS (sodium dodecyl sulphate) at 370C, and a wash in 1 X to 2 X SSC (20 X SSC=3.0 M NaCI/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCI, 1% SDS at 37°C, and a wash in 0.5 X to 1 X SSC at 55 to 600C.

The following are examples of sets of hybridization/wash conditions that may be used to clone orthologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 2 X SSC, 0. 1% SDS at 500C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 1 X SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50*C with washing in 0.5 X SSC, 0. 1% SDS at 50 0 C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1% SDS at 65 0

C.

"DNA shuffling" is a method to introduce mutations or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly. The DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule PF 56134 24 t' derived from at least one template DNA molecule. The shuffled DNA preferably en- O codes a variant polypeptide modified with respect to the polypeptide encoded by the template DNA, and may have an altered biological activity with respect to the polypeptide encoded by the template DNA.

S "Recombinant DNA molecule' is a combination of DNA sequences that are joined together using recombinant DNA technology and procedures used to join together DNA sequences as described, for example, in Sambrook et al., 1989.

O 10 The word "plant" refers to any plant, particularly to agronomically useful plants seed plants), and "plant cell" is a structural and physiological unit of the plant, which Scomprises a cell wall but may also refer to a protoplast. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, Sfor example, a plant tissue, or a plant organ differentiated into a structure that is present at any stage of a plant's development. Such structures include one or more plant organs including, but are not limited to, fruit, shoot, stem, leaf, flower petal, etc. Preferably, the term "plant" includes whole plants, shoot vegetative organs/structures (e.g.

leaves, stems and tubers), roots, flowers and floral organs/structures bracts, sepals, petals, stamens, carpels, anthers and ovules), seeds (including embryo, endosperm, and seed coat) and fruits (the mature ovary), plant tissues vascular tissue, ground tissue, and the like) and cells guard cells, egg cells, trichomes and the like), and progeny of same.

The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous. Included within the scope of the invention are all genera and species of higher and lower plants of the plant kingdom.

Included are furthermore the mature plants, seed, shoots and seedlings, and parts, propagation material (for example seeds and fruit) and cultures, for example cell cultures, derived therefrom. Preferred are plants and plant materials of the following plant families: Amaranthaceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Tetragoniaceae.

Annual, perennial, monocotyledonous and dicotyledonous plants are preferred host organisms for the generation of transgenic plants. The use of the recombination system, or method according to the invention is furthermore advantageous in all ornamental plants, forestry, fruit, or ornamental trees, flowers, cut flowers, shrubs or turf. Said plant may include but shall not be limited to bryophytes such as, for example, Hepaticae (hepaticas) and Musci (mosses); pteridophytes such as ferns, horsetail and clubmosses; gymnosperms such as conifers, cycads, ginkgo and Gnetaeae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms) and Euglenophyceae.

I

PF 56134 n Plants for the purposes of the invention may comprise the families of the Rosaceae 8 such as rose, Ericaceae such as rhododendrons and azaleas, Euphorbiaceae such as c poinsettias and croton, Caryophyllaceae such as pinks, Solanaceae such as petunias, SGesneriaceae such as African violet, Balsaminaceae such as touch-me-not, Orchidaceae such as orchids, Iridaceae such as gladioli, iris, freesia and crocus, Compositae such as marigold, Geraniaceae such as geraniums, Liliaceae such as Drachaena, Moraceae such as ficus, Araceae such as philodendron and many others.

The transgenic plants according to the invention are furthermore selected in particular from among dicotyledonous crop plants such as, for example, from the families of the Leguminosae such as pea, alfalfa and soybean; the family of the Umbelliferae, particularly the genus Daucus (very particularly the species carota (carrot)) and Apium (very particularly the species graveolens var. dulce (celery)) and many others; the family of Ithe Solanaceae, particularly the genus Lycopersicon, very particularly the species esculentum (tomato) and the genus Solanum, very particularly the species tuberosum (potato) and melongena (aubergine), tobacco and many others; and the genus Capsicum, very particularly the species annum (pepper) and many others; the family of the Leguminosae, particularly the genus Glycine, very particularly the species max (soybean) and many others; and the family of the Cruciferae, particularly the genus Brassica, very particularly the species napus (oilseed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli); and the genus Arabidopsis, very particularly the species thaliana and many others; the family of the Compositae, particularly the genus Lactuca, very particularly the species sativa (lettuce) and many others.

The transgenic plants according to the invention may be selected among monocotyledonous crop plants, such as, for example, cereals such as wheat, barley, sorghum and millet, rye, triticale, maize, rice or oats, and sugarcane. Further preferred are trees such as apple, pear, quince, plum, cherry, peach, nectarine, apricot, papaya, mango, and other woody species including coniferous and deciduous trees such as poplar, pine, sequoia, cedar, oak, etc. Especially preferred are Arabidopsis thaliana, Nicotiana tabacum, oilseed rape, soybean, corn (maize), wheat, linseed, potato and tagetes.

"Significant increase" is an increase that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater.

"Significantly less" means that the decrease is larger than the margin of error inherent in the measurement technique, preferably a decrease by about 2-fold or greater.

DETAILED DESCRIPTION OF THE INVENTION The present invention thus provides for isolated nucleic acid molecules comprising a plant nucleotide sequence that directs meristem-preferential or meristem-specific transcription of an operably linked nucleic acid fragment in a plant cell.

Specifically, the present invention provides transgenic expression cassettes for regulating meristem-preferential or meristem-specific expression in plants comprising

I

PF 56134 26 V i) at least one transcription regulating nucleotide sequence of a plant gene, said plant O gene selected from the group of genes described by the GenBank Arabidopsis C thaliana genome locii At2g02180, At5g54510, At2g26970, At2g01180, At3g45560, O At4g00580, At1g54480, or At4g11490, or the Arabidopsis thaliana genomic se- S 5 quences as described by SEQ ID NO: 35 or 36, or a functional equivalent thereof, and functionally linked thereto ii) at least one nucleic acid sequence which is heterologous in relation to said transcription regulating nucleotide sequence.

D 10 The term "meristem" in the context of the invention means the usually group of undifferentiated cells from which new tissues and organs are produced. Meristems are C characterized by active cell division. Meristems are plant tissues composed of dividing i cells and giving rise to organs such as leaves, flowers, xylem, phloem, roots. Meris- Items are regions of a plant in which cells are not fully differentiated and which are capable of repeated mitotic divisions. Most plants have apical meristems which give rise c to the primary tissues of plants. The main meristematic areas within the plant are the apical meristems of the terminal and lateral shoots, the vascular cambium, the root apex, and the marginal meristems (active during the growth of leaves). Lateral meristems exist near root and shoot tips causing vertical plant growth. Higher plants produce most organs post-embryonically, including stems, leaves and roots. These organs develop from meristems at the tip of the stem and the root that are called the shoot apical meristem (SAM) and the root apical meristem, respectively. In dicots, the SAM serves as source of pluripotent stem cells and plays a central role in shoot organ formation.

Meristem specific promoters are useful for regulation of expression of several genes in meristematic cells, especially in meristems of leaf axils and abscission zones of flowers, fruits, siliques or pods. Beside the more general applications described below, the meristem specific or preferential promoters of the invention are useful for one or more of the following applications: a) specific expression in shoot meristem of genes involved in regulating development.

Such genes include those involved in flowering, as well genes that protect against pathogens by encoding toxins (see US 5,880,330) b) expression of insect resistance or tolerance, herbicide resistance or tolerance, disease resistance or tolerance resistance to viruses or fungal pathogens), stress tolerance (increased salt tolerance) and improved food content or increased yield (see WO01/18211) c) expression of genes for reducing formation of lateral shoots particularly e.g. in tomato, tobacco, wine, cereals and lumber. Lateral shoots are sink organs and reduce the yield of main shoots. Wild or great branching systems are difficult to harvest with machines. Fruits on main and lateral sprouts are ripening to different time points.

This prevents a concurrent harvest. Undesired nods in lumber has to be removed (see W002/06487).

d) expression of genes like cell wall invertases to accelerate flowering resulting in an increase in seed yield. (Heyer AG et al. (2004) Plant J. 39 (2):161-169.

e) expression of genes controlling the transition to flowering, or genes to reduce losses due to pests and stresses damaging plant apical meristems.

r PF 56134 27 0 f) By inhibiting/over expression of proteins that modulate meristem development, and specifically increases meristem cell proliferation, enlargement of meristems in plants CN can be induced. This is useful for increasing meristem cell proliferation that causes 0 increased row number in maize and is useful for manipulating meristem growth, or- S 5 gan development, seed number, inflorescence development and arrangement, development and embryogenesis, to increase yield, health and stability of plants. (see WO 2001070987).

e) expression of transgenes that regulate cytokinin response. These approaches are useful for a variety of agricultural and commercial purposes including improving and O 10 enhancing photosynthesis, promoting cell proliferation, shoot meristem formation, promoting leaf developing, increasing crop yields, improving crop and ornamental Cl quality and reducing agricultural production costs (see WO 02/099079).

g) expression of genes encoding RALF polypeptides which are known to stimulate the Sgrowth of plant meristems (see. WO 01/60972). The yield of edible material from a crop plant and the yield of one or more desired chemical products produced C-i by a plant, depends, in part, on the size of the plant. The size of the plant is determined, at least in part, by the rate of growth of the plant meristems.

h) expression of enzymes to manipulate genomic DNA and ensure that said manipulation is transmitted to the next cell and to the progeny. For example, nucleases or recombinases can be fused to said promoters. In consequence, nucleases or recombinases are expressed in meristems and act on their target sequences in the genome. Nucleases or recombinases can induce recombination at their respective target site(s) for marker excision or site-specific integration. Various methods are known in the art for marker excision W003004659; W093/01283] and sitespecific integration W096/14408; WO 00/11155]).

i) Meristem specific promoters operably linked to target sequences are useful for conditional or regulated gene silencing in plants. Recombinase inversion or excision yields double-stranded RNA, which thereby functions to trigger endogenous gene silencing mechanism. By combination meristem specific promoters with recombinase systems, transcriptional stop fragments or introns and target sequences, gene silencing of virtually any target sequences may be modulated at any plant development stage or in any plant generation. This is especially useful, when genes responsible for gene silencing are down regulated to permit expression of particular transgenes at levels greater than permitted when gene silencing is activated (see. e.g., WO 2004/003180) i) Meristem specific promoters are useful to confer virus induced gene silencing across meristematic tissue for altering the phenotype of a plant which involves silencing the target gene. e.g. an unwanted trait in a plant (see CA 2297616).

"Meristem-specific transcription" in the context of this invention means the transcription of a nucleic acid sequence by a transcription regulating element in a way that transcription of said nucleic acid sequence in the meristem contribute to more than 90%, preferably more than 95%, more preferably more than 99% of the entire quantity of the RNA transcribed from said nucleic acid sequence in the entire plant during any of its developmental stage. The transcription regulating nucleotide sequences designated pSUH415, pSUH415L, pSUH415GB, pSUH416, pSUH416GB, pSUH431, pSUH431GB, pSUH417, pSUH417GB, and pSUH432 and their respective shorter and PF 56134 28 0 longer variants are considered to be meristem-specific transcription regulating nucleotide sequences.

0 "Meristem-preferential transcription" in the context of this invention means the tran- S 5 scription of a nucleic acid sequence by a transcription regulating element in a way that transcription of said nucleic acid sequence in the meristem contribute to more than preferably more than 70%, more preferably more than 80% of the entire quantity of the RNA transcribed from said nucleic acid sequence in the entire plant during any of its developmental stage. The transcription regulating nucleotide sequences designated I 10 pSUH413, pSUH413GB, pSUH438, pSUH438v, pSUH438GB, pSUH433, pSUH433GB, pSUH436, pSUH436S, and pSUH436GB and their respective shorter CN and longer variants are considered to be meristem-preferential transcription regulating nucleotide sequences.

Preferably a transcription regulating nucleotide sequence of the invention comprises at Sleast one promoter sequence of the respective gene a sequence localized upstream of the transcription start of the respective gene capable to induce transcription of the downstream sequences). The transcription regulating nucleotide sequence may comprise the promoter sequence of said genes but may further comprise other elements such as the 5'-untranslated sequence, enhancer, introns etc. Preferably, said promoter sequence directs meristem-preferential or meristem-specific transcription of an operably linked nucleic acid segment in a plant or plant cell a linked plant DNA comprising an open reading frame for a structural or regulatory gene. The following Table 1 illustrates the genes from which the promoters of the invention are preferably isolated, the function of said genes, the cDNA encoded by said genes, and the protein (ORF) encoded by said genes.

Table 1: Genes from which the promoters of the invention are preferably isolated, putative function of said genes, cDNA and the protein encoded by said genes.

Gene Locus Putative function Promoter mRNA locus ID Proteine ID SEQ ID cDNA SEQ ID Protein SEQ ID At2g02180 tobamovirus multiplication pro- SEQ ID NO: NM_126278 NP_027422 tein 3 (TOM3) 1, 2, 3 SEQ ID NO: 4 SEQ ID NO: At5g54510 auxin-responsive GH3 protein SEQ ID NO: NM_124831 NP_200262 6,7,8 SEQIDNO: 9 At2g26970 exonuclease family protein SEQ ID NO: NM_179759 NP_850090 11,12 SEQ ID NO: 13 SEQ ID NO: 14 At2g01180 putative phosphatidic acid SEQ ID NO: NM_201660 NP 973389 phosphatase 15, 16,17 SEQ ID NO: 18 SEQ ID NO: 19 At3g45560 zinc finger (C3HC4-type RING SEQ ID NO: NM_114425 NP_190142 finger) family 20, 21 SEQ ID NO: 22 SEQ ID NO: 23 At4g00580 COP1-interacting protein- SEQ ID NO: NM 116282 NP 191967 related 24, 25 SEQ ID NO: 26 SEQ ID NO: 27 At1g54480 leucine-rich repeat family pro- SEQ ID NO: NM 104326 NP 175850 tein 28 SEQ ID NO: 29 SEQ ID NO: At4g11490 serin/threonin kinase like pro- SEQ ID NO: NM_117220 NP 192888 tein 31, 32 SEQ ID NO: 33 SEQ ID NO: 34 no EST correlation SEQ ID NO: sequence is positioned down- 35, 36 stream and in opposite direction to ORF of gene At2g31160 r PF 56134 29 V Preferably the transcription regulating nucleotide sequence (or the functional equivalent thereof) is selected from the group of sequences consisting of N i) the sequences described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, O 21, 24, 25, 28, 31, 32, 35, and 36, ii) a fragment of at least 50 consecutive bases of a sequence under i) which has substantially the same promoter activity as the corresponding transcription regulating nucleotide sequence described by 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 28, 31, 32, 35, or 36; D iii) a nucleotide sequence having substantial similarity with a sequence identity of at least 40, 50, 60, to 70%, preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, 81% to 84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and C 99%) to a transcription regulating nucleotide sequence described by SEQ ID NO: 1,2,3,6,7,8, 11,12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, or 36; 0 15 iv) a nucleotide sequence capable of hybridizing (preferably under conditions equivac lent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 2 X SSC, 0. 1% SDS at 50°C (more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 0.5 X SSC, 0. 1% SDS at 50 0 C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 0.1 X SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1% SDS at 65 0 C) to a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 28, 31, 32, 35, or 36, or the complement thereof; v) a nucleotide sequence capable of hybridizing (preferably under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 2 X SSC, 0. 1% SDS at 50°C (more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1,mM EDTA at 50°C with washing in 0.5 X SSC, 0. 1% SDS at 500C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1% SDS at 65°C) to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, or 36, or the complement thereof; vi) a nucleotide sequence which is the complement or reverse complement of any of the previously mentioned nucleotide sequences under i) to v).

A functional equivalent of the transcription regulating nucleotide sequence can also be obtained or is obtainable from plant genomic DNA from a gene encoding a polypeptide which is substantially similar and preferably has at least 70%, preferably 80%, more preferably 90%, most preferably 95% amino acid sequence identity to a polypeptide PF 56134 Sencoded by an Arabidopsis thaliana gene comprising any one of SEQ ID NOs: 5, O 14, 19, 23, 27, 30, or 34, respectively, or a fragment of said transcription regulating nucleotide sequence which exhibits promoter activity in a meristem-preferential or meristem-specific fashion.

S The activity of a certain transcription regulating nucleotide sequence is considered equivalent if transcription is initiated preferentially or specifically in the same tissue (i.e.

meristematic tissue) than the original promoter. Such expression profile is preferably demonstrated using reporter genes operably linked to said transcription regulating nu- O 10 cleotide sequence. Preferred reporter genes (Schenborn 1999) in this context are green fluorescence protein (GFP) (Chui 1996; Leffel 1997), chloramphenicol trans- Sferase, luciferase (Millar 1992), 1-glucuronidase or P-galactosidase. Especially preferred is 1-glucuronidase (Jefferson 1987).

Beside this the transcription regulating activity of a function equivalent may vary from Sthe activity of its parent sequence, especially with respect to expression level. The expression level may be higher or lower than the expression level of the parent sequence. Both derivations may be advantageous depending on the nucleic acid sequence of interest to be expressed. Preferred are such functional equivalent sequences which in comparison with its parent sequence does not derivate from the expression level of said parent sequence by more than 50%, preferably 25%, more preferably 10% (as to be preferably judged by either mRNA expression or protein reporter gene) expression). Furthermore preferred are equivalent sequences which demonstrate an increased expression in comparison to its parent sequence, preferably an increase my at least 50%, more preferably by at least 100%, most preferably by at least 500%.

Preferably functional equivalent of the transcription regulating nucleotide sequence can be obtained or is obtainable from plant genomic DNA from a gene expressing a mRNA described by a cDNA which is substantially similar and preferably has at least preferably 80%, more preferably 90%, most preferably 95% sequence identity to a sequence described by any one of SEQ ID NOs: 4, 9, 13, 18, 22, 26, 29, or 33, respectively, or a fragment of said transcription regulating nucleotide sequence which exhibits promoter activity in a meristem-preferential or meristem-specific fashion.

Such functional equivalent of the transcription regulating nucleotide sequence may be obtained from other plant species by using the meristem-preferential or meristemspecific Arabidopsis promoter sequences described herein as probes to screen for homologous structural genes in other plants by hybridization under low, moderate or stringent hybridization conditions. Regions of the meristem-preferential or meristemspecific promoter sequences of the present invention which are conserved among species could also be used as PCR primers to amplify a segment from a species other than Arabidopsis, and that segment used as a hybridization probe (the latter approach permitting higher stringency screening) or in a transcription assay to determine promoter activity. Moreover, the meristem-preferential or meristem-specific promoter sequences could be employed to identify structurally related sequences in a database using computer algorithms.

PF 56134 31 SMore specifically, based on the Arabidopsis nucleic acid sequences of the present in- 8 vention, orthologs may be identified or isolated from the genome of any desired organism, preferably from another plant, according to well known techniques based on their Ssequence similarity to the Arabidopsis nucleic acid sequences, hybridization, PCR 5 or computer generated sequence comparisons. For example, all or a portion of a particular Arabidopsis nucleic acid sequence is used as a probe that selectively hybridizes to other gene sequences present in a population of cloned genomic DNA fragments or cDNA fragments genomic or cDNA libraries) from a chosen source organism. Further, suitable genomic and cDNA libraries may be prepared from any cell or tissue of an organism. Such techniques include hybridization screening of plated DNA libraries _(either plaques or colonies; see, Sambrook 1989) and amplification by PCR using oligonucleotide primers preferably corresponding to sequence domains conserved among related polypeptide or subsequences of the nucleotide sequences provided herein (see, Innis 1990). These methods are particularly well suited to the isolation of gene sequences from organisms closely related to the organism from which the probe sequence is derived. The application of these methods using the Arabidopsis sequences as probes is well suited for the isolation of gene sequences from any source organism, preferably other plant species. In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art.

In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 p, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the sequence of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989). In general, sequences that hybridize to the sequences disclosed herein will have at least 40% to 50%, about 60% to and even about 80% 85%, 90%, 95% to 98% or more identity with the disclosed sequences. That is, the sequence similarity of sequences may range, sharing at least about 40% to 50%, about 60% to 70%, and even about 80%, 85%, 90%, 95% to 98% sequence similarity.

The nucleic acid molecules of the invention can also be identified by, for example, a search of known databases for genes encoding polypeptides having a specified amino acid sequence identity or DNA having a specified nucleotide sequence identity. Methods of alignment of sequences for comparison are well known in the art and are described hereinabove.

Hence, the isolated nucleic acid molecules of the invention include the orthologs of the Arabidopsis sequences disclosed herein, the corresponding nucleotide sequences in organisms other than Arabidopsis, including, but not limited to, plants other than PF 56134 32 Arabidopsis, preferably dicotyledonous plants, Brassica napus, alfalfa, sunflower, soybean, cotton, peanut, tobacco or sugar beet, but also cereal plants such as corn, C1 wheat, rye, turfgrass, sorghum, millet, sugarcane, barley and banana. An orthologous O gene is a gene from a different species that encodes a product having the same or similar function, catalyzing the same reaction as a product encoded by a gene from a reference organism. Thus, an ortholog includes polypeptides having less than, 65% amino acid sequence identity, but which ortholog encodes a polypeptide having the same or similar function. Databases such GenBank may be employed to identify sequences related to the Arabidopsis sequences, orthologs in other dicoty- I 10 ledonous plants such as Brassica napus and others. Alternatively, recombinant DNA techniques such as hybridization or PCR may be employed to identify sequences re- Cl lated to the Arabidopsis sequences or to clone the equivalent sequences from different Arabidopsis DNAs.

In The transcription regulating nucleotide sequences of the invention or their functional equivalents can be obtained or isolated from any plant or non-plant source, or produced synthetically by purely chemical means. Preferred sources include, but are not limited to the plants defined in the DEFINITION section above.

Thus, another embodiment of the invention relates to a method for identifying and/or isolating a sequence with meristem-preferential or meristem-specific transcription regulating activity utilizing a nucleic acid sequence encoding a amino acid sequence as described by SEQ ID NO: 5, 10, 14, 19, 23, 27, 30, or 34 or a part thereof. Preferred are nucleic acid sequences described by SEQ ID NO: 4, 9, 13, 18, 22, 26, 29, or 33 or parts thereof. "Part" in this context means a nucleic acid sequence of at least 15 bases preferably at least 25 bases, more preferably at least 50 bases. The method can be based on (but is not limited to) the methods described above such as polymerase chain reaction, hybridization or database screening. Preferably, this method of the invention is based on a polymerase chain reaction, wherein said nucleic acid sequence or its part is utilized as oligonucleotide primer. The person skilled in the art is aware of several methods to amplify and isolate the promoter of a gene starting from part of its coding sequence (such as, for example, part of a cDNA). Such methods may include but are not limited to method such as inverse PCR ("iPCR") or "thermal asymmetric interlaced PCR" ("TAIL PCR").

Another embodiment of the invention is related to a method for providing a transgenic expression cassette for meristem-preferential or meristem-specific expression comprising the steps of: I. isolating of a meristem-preferential or meristem-specific transcription regulating nucleotide sequence utilizing at least one nucleic acid sequence or a part thereof, wherein said sequence is encoding a polypeptide described by SEQ ID NO: 5, 14, 19, 23, 27, 30, or 34, or a part of at least 15 bases thereof, and II. functionally linking said meristem-preferential or meristem-specific transcription regulating nucleotide sequence to another nucleotide sequence of interest, which is heterologous in relation to said meristem-preferential or meristem-specific transcription regulating nucleotide sequence.

PF 56134 33 SPreferably, the nucleic acid sequence employed for the isolation comprises at least base, preferably at least 25 bases, more preferably at least 50 bases of a sequence C' described by SEQ ID NO: 4, 9, 13, 18, 22, 26, 29, or 33. Preferably, the isolation of the U meristem-preferential or meristem-specific transcription regulating nucleotide sequence is realized by a polymerase chain reaction utilizing said nucleic acid sequence as a primer. The operable linkage can be realized by standard cloning method known in the art such as ligation-mediated cloning or recombination-mediated cloning.

Preferably, the transcription regulating nucleotide sequences and promoters of the in- ID 10 vention include a consecutive stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500, contiguous nucleotides, 40 to about 743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743, of any one of SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, Sand 36, or the promoter orthologs thereof, which include the minimal promoter region.

C' In a particular embodiment of the invention said consecutive stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500, contiguous nucleotides, 40 to about 743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743, has at least 75%, preferably 80%, more preferably 90% and most preferably 95%, nucleic acid sequence identity with a corresponding consecutive stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500, contiguous nucleotides, 40 to about 743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743, of any one of SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, and 36, or the promoter orthologs thereof, which include the minimal promoter region. The above defined stretch of contiguous nucleotides preferably comprises one or more promoter motifs selected from the group consisting of TATA box, GC-box, CAAT-box and a transcription start site.

Thetranscription regulating nucleotide sequences of the invention or their functional equivalents are capable of driving meristem-preferential or meristem-specific expression of a coding sequence in a target cell, particularly in a plant cell. The promoter sequences and methods disclosed herein are useful in regulating meristem-preferential or meristem-specific expression, respectively, of any heterologous nucleotide sequence in a host plant in order to vary the phenotype of that plant. These promoters can be used with combinations of enhancer, upstream elements, and/or activating sequences from the 5' flanking regions of plant expressible structural genes. Similarly the upstream element can be used in combination with various plant promoter sequences.

The transcription regulating nucleotide sequences and promoters of the invention are useful to modify the phenotype of a plant. Various changes in the phenotype of a transgenic plant are desirable, modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants. Alternatively, the results can be achieved by providing for a reduction of expression of one or more en- PF 56134 34 Sdogenous products, particularly enzymes or cofactors in the plant. These changes re- 8 suit in an alteration in the phenotype of the transformed plant.

SGenerally, the transcription regulating nucleotide sequences and promoters of the in- S 5 vention may be employed to express a nucleic acid segment that is operably linked to said promoter such as, for example, an open reading frame, or a portion thereof, an anti-sense sequence, a sequence encoding for a double-stranded RNA sequence, or a transgene in plants.

IND 10 An operable linkage may for example comprise an sequential arrangement of the transcription regulating nucleotide sequence of the invention (for example a sequence c as described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, or 36 with a nucleic acid sequence to be expressed, and optionally addi- Stional regulatory elements such as for example polyadenylation or transcription termination elements, enhancers, introns etc, in a way that the transcription regulating nucleotide sequence can fulfill its function in the process of expression the nucleic acid sequence of interest under the appropriate conditions, the term "appropriate conditions" mean preferably the presence of the expression cassette in a plant cell. Preferred are arrangements, in which the nucleic acid sequence of interest to be expressed is placed down-stream in 3'-direction) of the transcription regulating nucleotide sequence of the invention in a way, that both sequences are covalently linked. Optionally additional sequences may be inserted in-between the two sequences. Such sequences may be for example linker or multiple cloning sites. Furthermore, sequences can be inserted coding for parts of fusion proteins (in case a fusion protein of the protein encoded by the nucleic acid of interest is intended to be expressed). Preferably, the distance between the nucleic acid sequence of interest to be expressed and the transcription regulating nucleotide sequence of the invention is not more than 200 base pairs, preferably not more than 100 base pairs, more preferably no more than 50 base pairs.

An operable linkage in relation to any expression cassette or of the invention may be realized by various methods known in the art, comprising both in vitro and in vivo procedure. Thus, an expression cassette of the invention or an vector comprising such expression cassette may by realized using standard recombination and cloning techniques well known in the art (see Maniatis 1989; Silhavy 1984; Ausubel 1987).

An expression cassette may also be assembled by inserting a transcription regulating nucleotide sequence of the invention (for example a sequence as described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, or 36) into the plant genome. Such insertion will result in an operable linkage to a nucleic acid sequence of interest which as such already existed in the genome. By the insertion the nucleic acid of interest is expressed in a meristem-preferential or meristem-specific way due to the transcription regulating properties of the transcription regulating nucleotide sequence. The insertion may be directed or by chance. Preferably the insertion is directed and realized by for example homologous recombination. By this procedure a natural promoter may be exchanged against the transcription regulating nucleotide sequence of the invention, thereby modifying the expression profile of an endogenous gene. The transcription regulating nucleotide sequence may also be inserted in a way, r PF 56134 n that antisense mRNA of an endogenous gene is expressed, thereby inducing gene 8 silencing.

SSimilar, a nucleic acid sequence of interest to be expressed may by inserted into a plant genome comprising the transcription regulating nucleotide sequence in its natural genomic environment linked to its natural gene) in a way that the inserted sequence becomes operably linked to the transcription regulating nucleotide sequence, thereby forming an expression cassette of the invention.

ID 10 The open reading frame to be linked to the transcription regulating nucleotide se- _quence of the invention may be obtained from an insect resistance gene, a disease N resistance gene such as, for example, a bacterial disease resistance gene, a fungal disease resistance gene, a viral disease resistance gene, a nematode disease resis- Stance gene, a herbicide resistance gene, a gene affecting grain composition or quality, a nutrient utilization gene, a mycotoxin reduction gene, a male sterility gene, a selectable marker gene, a screenable marker gene, a negative selectable marker, a positive selectable marker, a gene affecting plant agronomic characteristics, yield, standability, and the like, or an environment or stress resistance gene, one or more genes that confer herbicide resistance or tolerance, insect resistance or tolerance, disease resistance or tolerance (viral, bacterial, fungal, oomycete, or nematode), stress tolerance or resistance (as exemplified by resistance or tolerance to drought, heat, chilling, freezing, excessive moisture, salt stress, or oxidative stress), increased yields, food content and makeup, physical appearance, male sterility, drydown, standability, prolificacy, starch properties or quantity, oil quantity and quality, amino acid or protein composition, and the like. By "resistant" is meant a plant which exhibits substantially no phenotypic changes as a consequence of agent administration, infection with a pathogen, or exposure to stress. By "tolerant" is meant a plant which, although it may exhibit some phenotypic changes as a consequence of infection, does not have a substantially decreased reproductive capacity or substantially altered metabolism.

Meristem-preferential or meristem-specific transcription regulating nucleotide sequences promoters) are useful for expressing a wide variety of genes including those which alter metabolic pathways, confer disease resistance, for protein production, antibody production, or to improve nutrient uptake and the like. Meristempreferential or meristem-specific transcription regulating nucleotide sequences promoters) may be modified so as to be regulatable, inducible. The genes and transcription regulating nucleotide sequences promoters) described hereinabove can be used to identify orthologous genes and their transcription regulating nucleotide sequences promoters) which are also likely expressed in a particular tissue and/or development manner. Moreover, the orthologous transcription regulating nucleotide sequences promoters) are useful to express linked open reading frames.

In addition, by aligning the transcription regulating nucleotide sequences promoters) of these orthologs, novel cis elements can be identified that are useful to generate synthetic transcription regulating nucleotide sequences promoters).

The expression regulating nucleotide sequences specified above may be optionally operably linked to other suitable regulatory sequences, a transcription terminator

I

PF 56134 36 Ssequence, operator, repressor binding site, transcription factor binding site and/or an enhancer.

The present invention further provides a recombinant vector containing the expression S 5 cassette of the invention, and host cells comprising the expression cassette or vector, comprising a plasmid. The expression cassette or vector may augment the genome of a transformed plant or may be maintained extra chromosomally. The expression cassette or vector of the invention may be present in the nucleus, chloroplast, mitochondria and/or plastid of the cells of the plant. Preferably, the expression cassette or ID 10 vector of the invention is comprised in the chromosomal DNA of the plant nucleus. The present invention also provides a transgenic plant prepared by this method, a seed r from such a plant and progeny plants from such a plant including hybrids and inbreds.

The expression cassette may be operatively linked to a structural gene, the open read- Sing frame thereof, or a portion thereof. The expression cassette may further comprise a Ti plasmid and be contained in an Agrobacterium tumefaciens cell; it may be carried on a microparticle, wherein the microparticle is suitable for ballistic transformation of a plant cell; or it may be contained in a plant cell or protoplast. Further, the expression cassette or vector can be contained in a transformed plant or cells thereof, and the plant may be a dicot or a monocot. In particular, the plant may be a dicotyledonous plant. Preferred transgenic plants are transgenic maize, soybean, barley, alfalfa, sunflower, canola, soybean, cotton, peanut, sorghum, tobacco, sugarbeet, rice, wheat, rye, turfgrass, millet, sugarcane, tomato, or potato.

The invention also provides a method of plant breeding, to prepare a crossed fertile transgenic plant. The method comprises crossing a fertile transgenic plant comprising a particular expression cassette of the invention with itself or with a second plant, one lacking the particular expression cassette, to prepare the seed of a crossed fertile transgenic plant comprising the particular expression cassette. The seed is then planted to obtain a crossed fertile transgenic plant. The plant may be a monocot or a dicot. In a particular embodiment, the plant is a dicotyledonous plant. The crossed fertile transgenic plant may have the particular expression cassette inherited through a female parent or through a male parent. The second plant may be an inbred plant. The crossed fertile transgenic may be a hybrid. Also included within the present invention are seeds of any of these crossed fertile transgenic plants.

The transcription regulating nucleotide sequences of the invention further comprise sequences which are complementary to one (hereinafter "test" sequence) which hybridizes under stringent conditions with a nucleic acid molecule as described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, or36 as well as RNA which is transcribed from the nucleic acid molecule. When the hybridization is performed under stringent conditions, either the test or nucleic acid molecule of invention is preferably supported, on a membrane or DNA chip. Thus, either a denatured test or nucleic acid molecule of the invention is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of, e.g., between 55 and 70 0 C, in double strength citrate buffered saline (SC) containing 0.1% SDS followed by rinsing of the support at the same temperature but with a buffer having a reduced SC concentration. Depending upon the degree of stringency required PF 56134 37 V)such reduced concentration buffers are typically single strength SC containing 0.1% O SDS, half strength SC containing 0.1% SDS and one-tenth strength SC containing N 0.1% SDS. More preferably hybridization is carried out under high stringency condi- Stions (as defined above).

S Virtually any DNA composition may be used for delivery to recipient plant cells, e.g., dicotyledonous cells, to ultimately produce fertile transgenic plants in accordance with the present invention. For example, DNA segments or fragments in the form of vectors and plasmids, or linear DNA segments or fragments, in some instances containing only O 10 the DNA element to be expressed in the plant, and the like, may be employed. The construction of vectors which may be employed in conjunction with the present invenr tion will be known to those of skill of the art in light of the present disclosure (see, e.g., Sambrook 1989; Gelvin 1990).

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial arti- Sficial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise the cDNA, gene or genes which one desires to introduce into the cells. These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even regulatory genes as desired. The DNA segment, fragment or gene chosen for cellular introduction will often encode a protein which will be expressed in the resultant recombinant cells, such as will result in a screenable or selectable trait and/or which will impart an improved phenotype to the regenerated plant.

However, this may not always be the case, and the present invention also encompasses transgenic plants incorporating non-expressed transgenes.

In certain embodiments, it is contemplated that one may wish to employ replicationcompetent viral vectors in monocot transformation. Such vectors include, for example, wheat dwarf virus (WDV) "shuttle" vectors, such as pW1-11 and PW1-GUS (Ugaki 1991). These vectors are capable of autonomous replication in maize cells as well as E. coli, and as such may provide increased sensitivity for detecting DNA delivered to transgenic cells. A replicating vector may also be useful for delivery of genes flanked by DNA sequences from transposable elements such as Ac, Ds, or Mu. It has been proposed (Laufs 1990) that transposition of these elements within the maize genome requires DNA replication. It is also contemplated that transposable elements would be useful for introducing DNA segments or fragments lacking elements necessary for selection and maintenance of the plasmid vector in bacteria, antibiotic resistance genes and origins of DNA replication. It is also proposed that use of a transposable element such as Ac, Ds, or Mu would actively promote integration of the desired DNA and hence increase the frequency of stably transformed cells. The use of a transposable element such as Ac, Ds, or Mu may actively promote integration of the DNA of interest and hence increase the frequency of stably transformed cells. Transposable elements may be useful to allow separation of genes of interest from elements necessary for selection and maintenance of a plasmid vector in bacteria or selection of a transformant. By use of a transposable element, desirable and undesirable DNA sequences may be transposed apart from each other in the genome, such that through genetic segregation in progeny, one may identify plants with either the desirable undesirable DNA sequences.

PF 56134 38 SThe nucleotide sequence of interest linked to one or more of the transcription regulating nucleotide sequences of the invention can, for example, code for a ribosomal RNA, C an antisense RNA or any other type of RNA that is not translated into protein. In an- U other preferred embodiment of the invention, said nucleotide sequence of interest is S 5 translated into a protein product. The transcription regulating nucleotide sequence and/or nucleotide sequence of interest linked thereto may be of homologous or heterologous origin with respect to the plant to be transformed. A recombinant DNA molecule useful for introduction into plant cells includes that which has been derived or isolated from any source, that may be subsequently characterized as to structure, size O 10 and/or function, chemically altered, and later introduced into plants. An example of a nucleotide sequence or segment of interest "derived" from a source, would be a nu- Scleotide sequence or segment that is identified as a useful fragment within a given organism, and which is then chemically synthesized in essentially pure form. An example Sof such a nucleotide sequence or segment of interest "isolated" from a source, would be nucleotide sequence or segment that is excised or removed from said source by Schemical means, by the use of restriction endonucleases, so that it can be further manipulated, amplified, for use in the invention, by the methodology of genetic engineering. Such a nucleotide sequence or segment is commonly referred to as "recombinant." Therefore a useful nucleotide sequence, segment or fragment of interest includes completely synthetic DNA, semi-synthetic DNA, DNA isolated from biological sources, and DNA derived from introduced RNA. Generally, the introduced DNA is not originally resident in the plant genotype which is the recipient of the DNA, but it is within the scope of the invention to isolate a gene from a given plant genotype, and to subsequently introduce multiple copies of the gene into the same genotype, to enhance production of a given gene product such as a storage protein or a protein that confers tolerance or resistance to water deficit.

The introduced recombinant DNA molecule includes but is not limited to, DNA from plant genes, and non-plant genes such as those from bacteria, yeasts, animals or viruses. The introduced DNA can include modified genes, portions of genes, or chimeric genes, including genes from the same or different genotype. The term "chimeric gene" or "chimeric DNA" is defined as a gene or DNA sequence or segment comprising at least two DNA sequences or segments from species which do not combine DNA under natural conditions, or which DNA sequences or segments are positioned or linked in a manner which does not normally occur in the native genome of untransformed plant.

The introduced recombinant DNA molecule used for transformation herein may be circular or linear, double-stranded or single-stranded. Generally, the DNA is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by regulatory sequences which promote the expression of the recombinant DNA present in the resultant plant. Generally, the introduced recombinant DNA molecule will be relatively small, less than about 30 kb to minimize any susceptibility to physical, chemical, or enzymatic degradation which is known to increase as the size of the nucleotide molecule increases. As noted above, the number of proteins, RNA transcripts or mixtures thereof which is introduced into the plant genome is preferably preselected PF 56134 39 Sand defined, from one to about 5-10 such products of the introduced DNA may be formed.

Two principal methods for the control of expression are known, viz.: overexpression S 5 and underexpression. Overexpression can be achieved by insertion of one or more than one extra copy of the selected gene. It is, however, not unknown for plants or their progeny, originally transformed with one or more than one extra copy of a nucleotide sequence, to exhibit the effects of underexpression as well as overexpression. For underexpression there are two principle methods which are commonly referred to in the N 10 art as "antisense downregulation" and "sense downregulation" (sense downregulation is also referred to as "cosuppression"). Generically these processes are referred to as S"gene silencing". Both of these methods lead to an inhibition of expression of the target gene.

Obtaining sufficient levels of transgene expression in the appropriate plant tissues is an Simportant aspect in the production of genetically engineered crops. Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed.

It is specifically contemplated by the inventors that one could mutagenize a promoter to potentially improve the utility of the elements for the expression of transgenes in plants.

The mutagenesis of these elements can be carried out at random and the mutagenized promoter sequences screened for activity in a trial-by-error procedure. Alternatively, particular sequences which provide the promoter with desirable expression characteristics, or the promoter with expression enhancement activity, could be identified and these or similar sequences introduced into the sequences via mutation. It is further contemplated that one could mutagenize these sequences in order to enhance their expression of transgenes in a particular species.

The means for mutagenizing a DNA segment encoding a promoter sequence of the current invention are well-known to those of skill in the art. As indicated, modifications to promoter or other regulatory element may be made by random, or site-specific mutagenesis procedures. The promoter and other regulatory element may be modified by altering their structure through the addition or deletion of one or more nucleotides from the sequence which encodes the corresponding unmodified sequences.

Mutagenesis may be performed in accordance with any of the techniques known in the art, such as, and not limited to, synthesizing an oligonucleotide having one or more mutations within the sequence of a particular regulatory region. In particular, sitespecific mutagenesis is a technique useful in the preparation of promoter mutants, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA se- PF 56134 r quence of the desired mutation, as well as a sufficient number of adjacent nucleotides, O to provide a primer sequence of sufficient size and sequence complexity to form a sta- C ble duplex on both sides of the deletion junction being traversed. Typically, a primer of 0 about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form.

I) 10 Typical vectors useful in site-directed mutagenesis include vectors such as the M13 Sphage. These phage are readily commercially available and their use is generally well Cknown to those skilled in the art. Double stranded plasmids also are routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.

0 In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the promoter. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and cells are selected which include recombinant vectors bearing the mutated sequence arrangement. Vector DNA can then be isolated from these cells and used for plant transformation. A genetic selection scheme was devised by Kunkel et al. (1987) to enrich for clones incorporating mutagenic oligonucleotides. Alternatively, the use of PCR with commercially available thermostable enzymes such as Taq polymerase may be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector. The PCRmediated mutagenesis procedures of Tomic et al. (1990) and Upender et al. (1995) provide two examples of such protocols. A PCR employing a thermostable ligase in addition to a thermostable polymerase also may be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector. The mutagenesis procedure described by Michael (1994) provides an example of one such protocol.

The preparation of sequence variants of the selected promoter-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of DNA sequences may be obtained. For example, recombinant vectors encoding the desired promoter sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

PF 56134 41 SAs used herein; the term "oligonucleotide directed mutagenesis procedure" refers to 8 template-dependent processes and vector-mediated propagation which result in an N increase in the concentration of a specific nucleic acid molecule relative to its initial Sconcentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed mutagenesis procedure" also is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template-dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary IND 10 base pairing (see, for example, Watson and Rarnstad, 1987). Typically, vector medi- _ated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U.S. Pat. No.

S4,237,224. A number of template dependent processes are available to amplify the target sequences of interest present in a sample, such methods being well known in the art and specifically disclosed herein below.

Where a clone comprising a promoter has been isolated in accordance with the instant invention, one may wish to delimit the essential promoter regions within the clone. One efficient, targeted means for preparing mutagenizing promoters relies upon the identification of putative regulatory elements within the promoter sequence. This can be initiated by comparison with promoter sequences known to be expressed in similar tissuespecific or developmentally unique manner. Sequences which are shared among promoters with similar expression patterns are likely candidates for the binding of transcription factors and are thus likely elements which confer expression patterns. Confirmation of these putative regulatory elements can be achieved by deletion analysis of each putative regulatory region followed by functional analysis of each deletion construct by assay of a reporter gene which is functionally attached to each construct. As such, once a starting promoter sequence is provided, any of a number of different deletion mutants of the starting promoter could be readily prepared.

Functionally equivalent fragments of a transcription regulating nucleotide sequence of the invention can also be obtained by removing or deleting non-essential sequences without deleting the essential one. Narrowing the transcription regulating nucleotide sequence to its essential, transcription mediating elements can be realized in vitro by trial-and-arrow deletion mutations, or in silico using promoter element search routines.

Regions essential for promoter activity often demonstrate clusters of certain, known promoter elements. Such analysis can be performed using available computer algorithms such as PLACE ("Plant Cis-acting Regulatory DNA Elements"; Higo 1999), the BIOBASE database "Transfac" (Biologische Datenbanken GmbH, Braunschweig; Wingender 2001) or the database PlantCARE (Lescot 2002).

Preferably, functional equivalent fragments of one of the transcription regulating nucleotide sequences of the invention comprises at least 100 base pairs, preferably, at least 200 base pairs, more preferably at least 500 base pairs of a transcription regulating nucleotide sequence as described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16,

I

PF 56134 42 S17, 20, 21, 24, 25, 28, 31, 32, 35, or 36 More preferably this fragment is starting from the 3'-end of the indicated sequences.

O Especially preferred are equivalent fragments of transcription regulating nucleotide sequences, which are obtained by deleting the region encoding the region of the mRNA, thus only providing the (untranscribed) promoter region. The untranslated region can be easily determined by methods known in the art (such as RACE analysis). Accordingly, some of the transcription regulating nucleotide sequences of the invention are equivalent fragments of other sequences (see Table 2 I 10 below).

Cl Table 2: Relationship of transcription regulating nucleotide sequences of the invention Transcription regulating Equivalent sequence Equivalent fragment In sequence SEQ ID NO: 1 (1630 bp) SEQ ID NO: 2 (1631 bp) SEQ ID NO: 3 (1200 bp) SSEQ ID NO: 6 (510 bp) SEQ ID NO: 7 (511 bp) SEQ ID NO: 8 (396 bp) SSEQ ID NO: 11 (2552 bp) SEQ ID NO: 12 (2552 bp) SEQ ID NO: 17 (2658 bp) -SEQ ID NO: 15 (2193 bp) SEQ ID NO: 16 (2192 bp) SEQ ID NO: 20 (2219 bp) SEQ ID NO: 21 (2218 bp) SEQ ID NO: 24 (2042 bp) SEQ ID NO: 25 (2044 bp) SEQ ID NO: 28 (2092 bp) SEQ ID NO: 31 (2512 bp) SEQ ID NO: 32 (2512 bp) SEQ ID NO: 35 (1854 bp) SEQ ID NO: 36 (1855 bp) As indicated above, deletion mutants, deletion mutants of the promoter of the invention also could be randomly prepared and then assayed. With this strategy, a series of constructs are prepared, each containing a different portion of the clone (a subclone), and these constructs are then screened for activity. A suitable means for screening for activity is to attach a deleted promoter or intron construct which contains a deleted segment to a selectable or screenable marker, and to isolate only those cells expressing the marker gene. In this way, a number of different, deleted promoter constructs are identified which still retain the desired, or even enhanced, activity. The smallest segment which is required for activity is thereby identified through comparison of the selected constructs. This segment may then be used for the construction of vectors for the expression of exogenous genes.

An expression cassette of the invention may comprise further regulatory elements. The term in this context is to be understood in the a broad meaning comprising all sequences which may influence construction or function of the expression cassette.

Regulatory elements may for example modify transcription and/or translation in prokaryotic or eukaryotic organism. In an preferred embodiment the expression cassette of the invention comprised downstream (in 3'-direction) of the nucleic acid sequence to be expressed a transcription termination sequence and optionally additional regulatory elements each operably liked to the nucleic acid sequence to be expressed (or the transcription regulating nucleotide sequence).

Additional regulatory elements may comprise additional promoter, minimal promoters, or promoter elements, which may modify the expression regulating properties. For PF 56134 43 0 example the expression may be made depending on certain stress factors such water stress, abscisin (Lam 1991) or heat stress (Schoffl 1989). Furthermore additional pro- C1 moters or promoter elements may be employed, which may realized expression in 0 other organisms (such as E.coli or Agrobacterium). Such regulatory elements can be S 5 find in the promoter sequences or bacteria such as amy and SP02 or in the promoter sequences of yeast or fungal promoters (such as ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, and ADH).

Furthermore, it is contemplated that promoters combining elements from more than one promoter may be useful. For example, US 5,491,288 discloses combining a Cauliflower Mosaic Virus promoter with a histone promoter. Thus, the elements from the CN promoters disclosed herein may be combined with elements from other promoters.

Promoters which are useful for plant transgene expression include those that are inin ducible, viral, synthetic, constitutive (Odell 1985), temporally regulated, spatially regulated, tissue-specific, and spatial-temporally regulated.

Where expression in specific tissues or organs is desired, tissue-specific promoters may be used. In contrast, where gene expression in response to a stimulus is desired, inducible promoters are the regulatory elements of choice. Where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. Additional regulatory sequences upstream and/or downstream from the core promoter sequence may be included in expression constructs of transformation vectors to bring about varying levels of expression of heterologous nucleotide sequences in a transgenic plant.

A variety of 5' and 3' transcriptional regulatory sequences are available for use in the present invention. Transcriptional terminators are responsible for the termination of transcription and correct mRNA polyadenylation. The 3' nontranslated regulatory DNA sequence preferably includes from about 50 to about 1,000, more preferably about 100 to about 1,000, nucleotide base pairs and contains plant transcriptional and translational termination sequences. Appropriate transcriptional terminators and those which are known to function in plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator, the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato, although other 3' elements known to those of skill in the art can also be employed. Alternatively, one also could use a gamma coixin, oleosin 3 or other terminator from the genus Coix.

Preferred 3' elements include those from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato.

As the DNA sequence between the transcription initiation site and the start of the coding sequence, the untranslated leader sequence, can influence gene expression, one may also wish to employ a particular leader sequence. Preferred leader sequences are contemplated to include those which include sequences predicted to direct opti- PF 56134 44 Smum expression of the attached gene, to include a preferred consensus leader O sequence which may increase or maintain mRNA stability and prevent inappropriate Sinitiation of translation. The choice of such sequences will be known to those of skill in Sthe art in light of the present disclosure. Sequences that are derived from genes that S 5 are highly expressed in plants will be most preferred.

Preferred regulatory elements also include the 5'-untranslated region, introns and the 3'-untranslated region of genes. Such sequences that have been found to enhance gene expression in transgenic plants include intron sequences from Adhl, 0 10 bronzel, actinl, actin 2 (WO 00/760067), or the sucrose synthase intron; see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)) C and viral leader sequences from TMV, MCMV and AMV; Gallie 1987). For exampie, a number of non-translated leader sequences derived from viruses are known to n enhance expression. Specifically, leader sequences from Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have Sbeen shown to be effective in enhancing expression Gallie 1987; Skuzeski 1990).

Other leaders known in the art include but are not limited to: Picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein 1989); Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus); MDMV leader (Maize Dwarf Mosaic Virus); Human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak 1991); Untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA (Jobling 1987; Tobacco mosaic virus leader (TMV), (Gallie 1989; and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel 1991. See also, Della- Cioppa 1987. Regulatory elements such as Adh intron 1 (Callis 1987), sucrose synthase intron (Vasil 1989) or TMV omega element (Gallie 1989), may further be included where desired. Especially preferred are the 5'-untranslated region, introns and the 3'untranslated region from the genes described by the GenBank Arabidopsis thaliana genome locii At2g02180, At5g54510, At2g26970, At2g01180, At3g45560, At4g00580, At1g54480, or At4g11490, or the Arabidopsis thaliana genomic sequences as described by SEQ ID NO: 35 or 36, or of functional equivalent thereof.

Additional preferred regulatory elements are enhancer sequences or polyadenylation sequences. Preferred polyadenylation sequences are those from plant genes or Agrobacterium T-DNA genes (such as for example the terminator sequences of the OCS (octopine synthase) or NOS (nopaline synthase) genes).

Examples of enhancers include elements from the CaMV 35S promoter, octopine synthase genes (Ellis el al., 1987), the rice actin I gene, the maize alcohol dehydrogenase gene (Callis 1987), the maize shrunken I gene (Vasil 1989), TMV Omega element (Gallie 1989) and promoters from non-plant eukaryotes yeast; Ma 1988). Vectors for use in accordance with the present invention may be constructed to include the ocs enhancer element. This element was first identified as a 16 bp palindromic enhancer from the octopine synthase (ocs) gene of ultilane (Ellis 1987), and is present in at least other promoters (Bouchez 1989). The use of an enhancer element, such as the ocs elements and particularly multiple copies of the element, will act to increase the level of transcription from adjacent promoters when applied in the context of plant transformation.

PF 56134 An expression cassette of the invention (or a vector derived therefrom) may comprise O additional functional elements, which are to be understood in the broad sense as all (Ni elements which influence construction, propagation, or function of an expression cassette or a vector or a transgenic organism comprising them. Such functional elements d) 5 may include origin of replications (to allow replication in bacteria; for the ORI of pBR322 or the P15A ori; Sambrook 1989), or elements required for Agrobacterium T- DNA transfer (such as for example the left and/or rights border of the T-DNA).

Ultimately, the most desirable DNA segments for introduction into, for example, a dicot IsO 10 genome, may be homologous genes or gene families which encode a desired trait increased yield per acre) and which are introduced under the control of novel Spromoters or enhancers, etc., or perhaps even homologous or tissue specific root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- or leaf-specific) promoters or

I

n control elements. Indeed, it is envisioned that a particular use of the present invention will be the expression of a gene in a meristem-preferential or meristem-specific manner.

Additionally, vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transgenic plant or in directing a protein to the extracellular environment. This will generally be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resultant transit or signal peptide will transport the protein to a particular intracellular or extracellular destination, respectively, and will then be posttranslationally removed. Transit or signal peptides act by facilitating the transport of proteins through intracellular membranes, vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane.

A particular example of such a use concerns the direction of a herbicide resistance gene, such as the EPSPS gene, to a particular organelle such as the chloroplast rather than to the cytoplasm. This is exemplified by the use of the rbcs transit peptide which confers plastid-specific targeting of proteins. In addition, it is proposed that it may be desirable to target certain genes responsible for male sterility to the mitochondria, or to target certain genes for resistance to phytopathogenic organisms to the extracellular spaces, or to target proteins to the vacuole.

By facilitating the transport of the protein into compartments inside and outside the cell, these sequences may increase the accumulation of gene product protecting them from proteolytic degradation. These sequences also allow for additional mRNA sequences from highly expressed genes to be attached to the coding sequence of the genes.

Since mRNA being translated by ribosomes is more stable than naked mRNA, the presence of translatable mRNA in front of the gene may increase the overall stability of the mRNA transcript from the gene and thereby increase synthesis of the gene product. Since transit and signal sequences are usually post-translationally removed from the initial translation product, the use of these sequences allows for the addition of extra translated sequences that may not appear on the final polypeptide. Targeting of

I

PF 56134 46 Scertain proteins may be desirable in order to enhance the stability of the protein (US O 5,545,818).

SIt may be useful to target DNA itself within a cell. For example, it may be useful to target introduced DNA to the nucleus as this may increase the frequency of transformation. Within the nucleus itself it would be useful to target a gene in order to achieve site specific integration. For example, it would be useful to have an gene introduced through transformation replace an existing gene in the cell. Other elements include those that can be regulated by endogenous or exogenous agents, by zinc finger O 10 proteins, including naturally occurring zinc finger proteins or chimeric zinc finger pro- _teins (see, US 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO C' 98/53057; WO 98/53058; WO 00/23464; WO 95/19431; and WO 98/54311) or myb-like transcription factors. For example, a chimeric zinc finger protein may include amino In acid sequences which bind to a specific DNA sequence (the zinc finger) and amino acid sequences that activate GAL 4 sequences) or repress the transcription of Sthe sequences linked to the specific DNA sequence.

It is one of the objects of the present invention to provide recombinant DNA molecules comprising a nucleotide sequence according to the invention operably linked to a nucleotide segment of interest.

A nucleotide segment of interest is reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest changes, and as developing nations open up world markets, new crops and technologies will also emerge. In addition, as the understanding of agronomic traits and characteristics such as yield and heterosis increase, the choice of genes for transformation will change accordingly. General categories of nucleotides of interest include, for example, genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest include, generally, those involved in starch, oil, carbohydrate, or nutrient metabolism, as well as those affecting kernel size, sucrose loading, zinc finger proteins, see, US 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO 98/53058; WO 00/23464; WO 95/19431; and WO 98/54311, and the like.

One skilled in the art recognizes that the expression level and regulation of a transgene in a plant can vary significantly from line to line. Thus, one has to test several lines to find one with the desired expression level and regulation. Once a line is identified with the desired regulation specificity of a chimeric Cre transgene, it can be crossed with lines carrying different inactive replicons or inactive transgene for activation.

Other sequences which may be linked to the gene of interest which encodes a polypeptide are those which can target to a specific organelle, to the mitochondria, nucleus, or plastid, within the plant cell. Targeting can be achieved by providing the polypeptide with an appropriate targeting peptide sequence, such as a secretory signal PF 56134 47 Speptide (for secretion or cell wall or membrane targeting, a plastid transit peptide, a O chloroplast transit peptide, the chlorophyll a/b binding protein, a mitochondrial Starget peptide, a vacuole targeting peptide, or a nuclear targeting peptide, and the like.

For example, the small subunit of ribulose bisphosphate carboxylase transit peptide, the EPSPS transit peptide or the dihydrodipicolinic acid synthase transit peptide may be used. For examples of plastid organelle targeting sequences (see WO 00/12732).

Plastids are a class of plant organelles derived from proplastids and include chloroplasts, leucoplasts, amyloplasts, and chromoplasts. The plastids are major sites of biosynthesis in plants. In addition to photosynthesis in the chloroplast, plastids are also N 10 sites of lipid biosynthesis, nitrate reduction to ammonium, and starch storage. And while plastids contain their own circular, genome, most of the proteins localized to the Splastids are encoded by the nuclear genome and are imported into the organelle from the cytoplasm.

In 0 15 Transgenes used with the present invention will often be genes that direct the expres- Ssion of a particular protein or polypeptide product, but they may also be nonexpressible DNA segments, transposons such as Ds that do no direct their own transposition. As used herein, an "expressible gene" is any gene that is capable of being transcribed into RNA mRNA, antisense RNA, etc.) or translated into a protein, expressed as a trait of interest, or the like, etc., and is not limited to selectable, screenable or non-selectable marker genes. The invention also contemplates that, where both an expressible gene that is not necessarily a marker gene is employed in combination with a marker gene, one may employ the separate genes on either the same or different DNA segments for transformation. In the latter case, the different vectors are delivered concurrently to recipient cells to maximize cotransformation.

The choice of the particular DNA segments to be delivered to the recipient cells will often depend on the purpose of the transformation. One of the major purposes of transformation of crop plants is to add some commercially desirable, agronomically important traits to the plant. Such traits include, but are not limited to, herbicide resistance or tolerance; insect resistance or tolerance; disease resistance or tolerance (viral, bacterial, fungal, nematode); stress tolerance and/or resistance, as exemplified by resistance or tolerance to drought, heat, chilling, freezing, excessive moisture, salt stress; oxidative stress; increased yields; food content and makeup; physical appearance; male sterility; drydown; standability; prolificacy; starch properties; oil quantity and quality; and the like. One may desire to incorporate one or more genes conferring any such desirable trait or traits, such as, for example, a gene or genes encoding pathogen resistance.

In certain embodiments, the present invention contemplates the transformation of a recipient cell with more than one advantageous transgene. Two or more transgenes can be supplied in a single transformation event using either distinct transgeneencoding vectors, or using a single vector incorporating two or more gene coding sequences. For example, plasmids bearing the bar and aroA expression units in either convergent, divergent, or colinear orientation, are considered to be particularly useful.

Further preferred combinations are those of an insect resistance gene, such as a Bt gene, along with a protease inhibitor gene such as pinll, or the use of bar in combina- PF 56134 48 Stion with either of the above genes. Of course, any two or more transgenes of any de- 8 scription, such as those conferring herbicide, insect, disease (viral, bacterial, fungal, nematode) or drought resistance, male sterility, drydown, standability, prolificacy, starch properties, oil quantity and quality, or those increasing yield or nutritional quality may be employed as desired.

1. Exemplary Transgenes 1.1. Herbicide Resistance The genes encoding phosphinothricin acetyltransferase (bar and pat), glyphosate tolerant EPSP synthase genes, the glyphosate degradative enzyme gene gox encoding glyphosate oxidoreductase, deh (encoding a dehalogenase enzyme that inactivates Sdalapon), herbicide resistant sulfonylurea and imidazolinone) acetolactate syn- Cthase, and bxn genes (encoding a nitrilase enzyme that degrades bromoxynil) are good Sexamples of herbicide resistant genes for use in transformation. The bar and pat genes code for an enzyme, phosphinothricin acetyltransferase (PAT), which inactivates the CN herbicide phosphinothricin and prevents this compound from inhibiting glutamine synthetase enzymes. The enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is normally inhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate). However, genes are known that encode glyphosate-resistant EPSP Synthase enzymes. The deh gene encodes the enzyme dalapon dehalogenase and confers resistance to the herbicide dalapon. The bxn gene codes for a specific nitrilase enzyme that converts bromoxynil to a non-herbicidal degradation product.

1.2 Insect Resistance An important aspect of the present invention concerns the introduction of insect resistance-conferring genes into plants. Potential insect resistance genes which can be introduced include Bacillus thuringiensis crystal toxin genes or Bt genes (Watrud 1985).

Bt genes may provide resistance to lepidopteran or coleopteran pests such as European Corn Borer (ECB) and corn rootworm (CRW). Preferred Bt toxin genes for use in such embodiments include the CrylA(b) and CrylA(c) genes. Endotoxin genes from other species of B. thuringiensis which affect insect growth or development may also be employed in this regard. Protease inhibitors may also provide insect resistance (Johnson 1989), and will thus have utility in plant transformation. The use of a protease inhibitor II gene, pinll, from tomato or potato is envisioned to be particularly useful.

Even more advantageous is the use of a pinll gene in combination with a Bt toxin gene, the combined effect of which has been discovered by the present inventors to produce synergistic insecticidal activity. Other genes which encode inhibitors of the insects' digestive system, or those that encode enzymes or co-factors that facilitate the production of inhibitors, may also be useful. This group may be exemplified by cystatin and amylase inhibitors, such as those from wheat and barley.

Also, genes encoding lectins may confer additional or alternative insecticide properties.

Lectins (originally termed phytohemagglutinins) are multivalent carbohydrate-binding proteins which have the ability to agglutinate red blood cells from a range of species.

Lectins have been identified recently as insecticidal agents with activity against weevils, ECB and rootworm (Murdock 1990; Czapla Lang, 1990). Lectin genes contem- PF 56134 49 plated to be useful include, for example, barley and wheat germ agglutinin (WGA) and o rice lectins (Gatehouse 1984), with WGA being preferred.

SGenes controlling the production of large or small polypeptides active against insects when introduced into the insect pests, such as, lytic peptides, peptide hormones and toxins and venoms, form another aspect of the invention. For example, it is contemplated, that the expression of juvenile hormone esterase, directed towards specific insect pests, may also result in insecticidal activity, or perhaps cause cessation of metamorphosis (Hammock 1990).

INO _Transgenic plants expressing genes which encode enzymes that affect the integrity of the insect cuticle form yet another aspect of the invention. Such genes include those encoding, chitinase, proteases, lipases and also genes for the production of nik- Skomycin, a compound that inhibits chitin synthesis, the introduction of any of which is contemplated to produce insect resistant maize plants. Genes that code for activities that affect insect molting, such those affecting the production of ecdysteroid UDPglucosyl transferase, also fall within the scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compounds that reduce the nutritional quality of the host plant to insect pests are also encompassed by the present invention. It may be possible, for instance, to confer insecticidal activity on a plant by altering its sterol composition. Sterols are obtained by insects from their diet and are used for hormone synthesis and membrane stability. Therefore alterations in plant sterol composition by expression of novel genes, those that directly promote the production of undesirable sterols or those that convert desirable sterols into undesirable forms, could have a negative effect on insect growth and/or development and hence endow the plant with insecticidal activity. Lipoxygenases are naturally occurring plant enzymes that have been shown to exhibit anti-nutritional effects on insects and to reduce the nutritional quality of their diet. Therefore, further embodiments of the invention concern transgenic plants with enhanced lipoxygenase activity which may be resistant to insect feeding.

The present invention also provides methods and compositions by which to achieve qualitative or quantitative changes in plant secondary metabolites. One example concerns transforming plants to produce DIMBOA which, it is contemplated, will confer resistance to European corn borer, rootworm and several other maize insect pests.

Candidate genes that are particularly considered for use in this regard include those genes at the bx locus known to be involved in the synthetic DIMBOA pathway (Dunn 1981). The introduction of genes that can regulate the production of maysin, and genes involved in the production of dhurrin in sorghum, is also contemplated to be of use in facilitating resistance to earworm and rootworm, respectively.

Tripsacum dactyloides is a species of grass that is resistant to certain insects, including corn root worm. It is anticipated that genes encoding proteins that are toxic to insects or are involved in the biosynthesis of compounds toxic to insects will be isolated from Tripsacum and that these novel genes will be useful in conferring resistance to insects.

I PF 56134 l It is known that the basis of insect resistance in Tripsacum is genetic, because said resistance has been transferred to Zea mays via sexual crosses (Branson Guss, CN 1972).

Further genes encoding proteins characterized as having potential insecticidal activity may also be used as transgenes in accordance herewith. Such genes include, for example, the cowpea trypsin inhibitor (CpTI; Hilder 1987) which may be used as a rootworm deterrent; genes encoding avermectin (Campbell 1989; Ikeda 1987) which may prove particularly useful as a corn rootworm deterrent; ribosome inactivating protein 0 10 genes; and even genes that regulate plant structures. Transgenic maize including antiinsect antibody genes and genes that code for enzymes that can covert a non-toxic Cl insecticide (pro-insecticide) applied to the outside of the plant into an insecticide inside the plant are also contemplated.

1.3 Environment or Stress Resistance C Improvement of a plant's ability to tolerate various environmental stresses such as, but not limited to, drought, excess moisture, chilling, freezing, high temperature, salt, and oxidative stress, can also be effected through expression of heterologous, or overexpression of homologous genes. Benefits may be realized in terms of increased resistance to freezing temperatures through the introduction of an "antifreeze" protein such as that of the Winter Flounder (Cutler 1989) or synthetic gene derivatives thereof. Improved chilling tolerance may also be conferred through increased expression of glycerol-3-phosphate acetyltransferase in chloroplasts (Murata 1992; Wolter 1992). Resistance to oxidative stress (often exacerbated by conditions such as chilling temperatures in combination with high light intensities) can be conferred by expression of superoxide dismutase (Gupta 1993), and may be improved by glutathione reductase (Bowler 1992). Such strategies may allow for tolerance to freezing in newly emerged fields as well as extending later maturity higher yielding varieties to earlier relative maturity zones.

Expression of novel genes that favorably effect plant water content, total water potential, osmotic potential, and turgor can enhance the ability of the plant to tolerate drought. As used herein, the terms "drought resistance" and "drought tolerance" are used to refer to a plants increased resistance or tolerance to stress induced by a reduction in water availability, as compared to normal circumstances, and the ability of the plant to function and survive in lower-water environments, and perform in a relatively superior manner. In this aspect of the invention it is proposed, for example, that the expression of a gene encoding the biosynthesis of osmotically-active solutes can impart protection against drought. Within this class of genes are DNAs encoding mannitol dehydrogenase (Lee and Saier, 1982) and trehalose-6-phosphate synthase (Kaasen 1992). Through the subsequent action of native phosphatases in the cell or by the introduction and coexpression of a specific phosphatase, these introduced genes will result in the accumulation of either mannitol or trehalose, respectively, both of which have been well documented as protective compounds able to mitigate the effects of stress. Mannitol accumulation in transgenic tobacco has been verified and preliminary results indicate that plants expressing high levels of this metabolite are able to tolerate an applied osmotic stress (Tarczynski 1992).

PF 56134 51 t Similarly, the efficacy of other metabolites in protecting either enzyme function (e.g.

Salanopine or propionic acid) or membrane integrity alanopine) has been docu- Smented (Loomis 1989), and therefore expression of gene encoding the biosynthesis of Sthese compounds can confer drought resistance in a manner similar to or complimentary to mannitol. Other examples of naturally occurring metabolites that are osmotically active and/or provide some direct protective effect during drought and/or desiccation include sugars and sugar derivatives such as fructose, erythritol (Coxson 1992), sorbitol, dulcitol (Karsten 1992), glucosylglycerol (Reed 1984; Erdmann 1992), sucrose, stachyose (Koster Leopold 1988; Blackman 1992), ononitol and pinitol (Vernon 1O 10 Bohnert 1992), and raffinose (Bernal-Lugo Leopold 1992). Other osmotically active Ssolutes which are not sugars include, but are not limited to, proline and glycine-betaine N (Wyn-Jones and Storey, 1981). Continued canopy growth and increased reproductive fitness during times of stress can be augmented by introduction and expression of In genes such as those controlling the osmotically active compounds discussed above and other such compounds, as represented in one exemplary embodiment by the enzyme myoinositol 0-methyltransferase.

It is contemplated that the expression of specific proteins may also increase drought tolerance. Three classes of Late Embryogenic Proteins have been assigned based on structural similarities (see Dure 1989). All three classes of these proteins have been demonstrated in maturing desiccating) seeds. Within these 3 types of proteins, the Type-il (dehydrin-type) have generally been implicated in drought and/or desiccation tolerance in vegetative plant parts Mundy and Chua, 1988; Piatkowski 1990; Yamaguchi-Shinozaki 1992). Recently, expression of a Type-Ill LEA (HVA-1) in tobacco was found to influence plant height, maturity and drought tolerance (Fitzpatrick, 1993).

Expression of structural genes from all three groups may therefore confer drought tolerance. Other types of proteins induced during water stress include thiol proteases, aldolases and transmembrane transporters (Guerrero 1990), which may confer various protective and/or repair-type functions during drought stress. The expression of a gene that effects lipid biosynthesis and hence membrane composition can also be useful in conferring drought resistance on the plant.

Many genes that improve drought resistance have complementary modes of action.

Thus, combinations of these genes might have additive and/or synergistic effects in improving drought resistance in maize. Many of these genes also improve freezing tolerance (or resistance); the physical stresses incurred during freezing and drought are similar in nature and may be mitigated in similar fashion. Benefit may be conferred via constitutive or tissue-specific expression of these genes, but the preferred means of expressing these novel genes may be through the use of a turgor-induced promoter (such as the promoters for the turgor-induced genes described in Guerrero et al. 1990 and Shagan 1993). Spatial and temporal expression patterns of these genes may enable maize to better withstand stress.

Expression of genes that are involved with specific morphological traits that allow for increased water extractions from drying soil would be of benefit. For example, introduction and expression of genes that alter root characteristics may enhance water uptake.

Expression of genes that enhance reproductive fitness during times of stress would be PF 56134 52 r of significant value. For example, expression of DNAs that improve the synchrony of O pollen shed and receptiveness of the female flower parts, silks, would be of benefit.

In addition, expression of genes that minimize kernel abortion during times of stress Swould increase the amount of grain to be harvested and hence be of value. Regulation of cytokinin levels in monocots, such as maize, by introduction and expression of an isopentenyl transferase gene with appropriate regulatory sequences can improve monocot stress resistance and yield (Gan 1995).

Given the overall role of water in determining yield, it is contemplated that enabling \0 10 plants to utilize water more efficiently, through the introduction and expression of novel genes, will improve overall performance even when soil water availability is not limiting.

C, By introducing genes that improve the ability of plants to maximize water usage across a full range of stresses relating to water availability, yield stability or consistency of n yield performance may be realized.

SImproved protection of the plant to abiotic stress factors such as drought, heat or chill, can also be achieved for example by overexpressing antifreeze polypeptides from Myoxocephalus Scorpius (WO 00/00512), Myoxocephalus octodecemspinosus, the Arabidopsis thaliana transcription activator CBF1, glutamate dehydrogenases (WO 97/12983, WO 98/11240), calcium-dependent protein kinase genes (WO 98/26045), calcineurins (WO 99/05902), casein kinase from yeast (WO 02/052012), farnesyltransferases (WO 99/06580; Pei ZM et al. (1998) Science 282:287-290), ferritin (Deak M et al. (1999) Nature Biotechnology 17:192-196), oxalate oxidase (WO 99/04013; Dunwell JM (1998) Biotechn Genet Eng Rev 15:1-32), DREB1A factor ("dehydration response element B 1A"; Kasuga M et al. (1999) Nature Biotech 17:276-286), genes of mannitol or trehalose synthesis such as trehalose-phosphate synthase or trehalose-phosphate phosphatase (WO 97/42326) or by inhibiting genes such as trehalase (WO 97/50561).

1.4 Disease Resistance It is proposed that increased resistance to diseases may be realized through introduction of genes into plants period. It is possible to produce resistance to diseases caused, by viruses, bacteria, fungi, root pathogens, insects and nematodes. It is also contemplated that control of mycotoxin producing organisms may be realized through expression of introduced genes.

Resistance to viruses may be produced through expression of novel genes. For example, it has been demonstrated that expression of a viral coat protein in a transgenic plant can impart resistance to infection of the plant by that virus and perhaps other closely related viruses (Cuozzo 1988, Hemenway 1988, Abel 1986). It is contemplated that expression of antisense genes targeted at essential viral functions may impart resistance to said virus. For example, an antisense gene targeted at the gene responsible for replication of viral nucleic acid may inhibit said replication and lead to resistance to the virus. It is believed that interference with other viral functions through the use of antisense genes may also increase resistance to viruses. Further it is proposed that it may be possible to achieve resistance to viruses through other approaches, including, but not limited to the use of satellite viruses.

PF 56134 53 i It is proposed that increased resistance to diseases caused by bacteria and fungi may Sbe realized through introduction of novel genes. It is contemplated that genes encoding N so-called "peptide antibiotics," pathogenesis related (PR) proteins, toxin resistance, O and proteins affecting host-pathogen interactions such as morphological characteristics S 5 will be useful. Peptide antibiotics are polypeptide sequences which are inhibitory to growth of bacteria and other microorganisms. For example, the classes of peptides referred to as cecropins and magainins inhibit growth of many species of bacteria and fungi. It is proposed that expression of PR proteins in plants may be useful in conferring resistance to bacterial disease. These genes are induced following pathogen at- D 10 tack on a host plant and have been divided into at least five classes of proteins (Bol 1990). Included amongst the PR proteins are beta-1,3-glucanases, chitinases, and osmotin and other proteins that are believed to function in plant resistance to disease organisms. Other genes have been identified that have antifungal properties, UDA I(stinging nettle lectin) and hevein (Broakgert 1989; Barkai-Golan 1978). It is known that 0 15 certain plant diseases are caused by the production of phytotoxins. Resistance to these Sdiseases could be achieved through expression of a novel gene that encodes an enzyme capable of degrading or otherwise inactivating the phytotoxin. Expression novel genes that alter the interactions between the host plant and pathogen may be useful in reducing the ability the disease organism to invade the tissues of the host plant, e.g., an increase in the waxiness of the leaf cuticle or other morphological characteristics.

Plant parasitic nematodes are a cause of disease in many plants. It is proposed that it would be possible to make the plant resistant to these organisms through the expression of novel genes. It is anticipated that control of nematode infestations would be accomplished by altering the ability of the nematode to recognize or attach to a host plant and/or enabling the plant to produce nematicidal compounds, including but not limited to proteins.

Furthermore, a resistance to fungi, insects, nematodes and diseases, can be achieved by by targeted accumulation of certain metabolites or proteins. Such proteins include but are not limited to glucosinolates (defense against herbivores), chitinases or glucanases and other enzymes which destroy the cell wall of parasites, ribosomeinactivating proteins (RIPs) and other proteins of the plant resistance and stress reaction as are induced when plants are wounded or attacked by microbes, or chemically, by, for example, salicylic acid, jasmonic acid or ethylene, or lysozymes from nonplant sources such as, for example, T4-lysozyme or lysozyme from a variety of mammals, insecticidal proteins such as Bacillus thuringiensis endotoxin, a-amylase inhibitor or protease inhibitors (cowpea trypsin inhibitor), lectins such as wheatgerm agglutinin, RNAses or ribozymes. Further examples are nucleic acids which encode the Trichoderma harzianum chit42 endochitinase (GenBank Acc. No.: S78423) or the Nhydroxylating, multi-functional cytochrome P-450 (CYP79) protein from Sorghum bicolor (GenBank Acc. No.: U32624), or functional equivalents of these. The accumulation of glucosinolates as protection from pests (Rask L et al. (2000) Plant Mol Biol 42:93-113; Menard R et al. (1999) Phytochemistry 52:29-35), the expression of Bacillus thuringiensis endotoxins (Vaeck et al. (1987) Nature 328:33-37) or the protection against attack by fungi, by expression of chitinases, for example from beans (Broglie et al. (1991) Science 254:1194-1197), is advantageous. Resistance to pests such as, for PF 56134 54 Sexample, the rice pest Nilaparvata lugens in rice plants can be achieved by expressing 8 the snowdrop (Galanthus nivalis) lectin agglutinin (Rao et al. (1998) Plant J 15(4):469- S77).The expression of synthetic crylA(b) and crylA(c) genes, which encode lepidopteraspecific Bacillus thuringiensis D-endotoxins can bring about a resistance to insect pests S 5 in various plants (Goyal RK et al. (2000) Crop Protection 19(5):307-312). Further target genes which are suitable for pathogen defense comprise "polygalacturonase-inhibiting protein". (PGIP), thaumatine, invertase and antimicrobial peptides such as lactoferrin (Lee TJ et al. (2002) J Amer Soc Horticult Sci 127(2):158-164).

1.5 Plant Agronomic Characteristics _Two of the factors determining where plants can be grown are the average daily temperature during the growing season and the length of time between frosts. Within the areas where it is possible to grow a particular plant, there are varying limitations on the Smaximal time it is allowed to grow to maturity and be harvested. The plant to be grown in a particular area is selected for its ability to mature and dry down to harvestable moisture content within the required period of time with maximum possible yield. Therefore, plant of varying maturities are developed for different growing locations. Apart from the need to dry down sufficiently to permit harvest is the desirability of having maximal drying take place in the field to minimize the amount of energy required for additional drying post-harvest. Also the more readily the grain can dry down, the more time there is available for growth and kernel fill. Genes that influence maturity and/or dry down can be identified and introduced into plant lines using transformation techniques to create new varieties adapted to different growing locations or the same growing location but having improved yield to moisture ratio at harvest. Expression of genes that are involved in regulation of plant development may be especially useful, the liguleless and rough sheath genes that have been identified in plants.

Genes may be introduced into plants that would improve standability and other plant growth characteristics. For example, expression of novel genes which confer stronger stalks, improved root systems, or prevent or reduce ear droppage would be of great value to the corn farmer. Introduction and expression of genes that increase the total amount of photoassimilate available by, for example, increasing light distribution and/or interception would be advantageous. In addition the expression of genes that increase the efficiency of photosynthesis and/or the leaf canopy would further increase gains in productivity. Such approaches would allow for increased plant populations in the field.

Delay of late season vegetative senescence would increase the flow of assimilate into the grain and thus increase yield. Overexpression of genes within plants that are associated with "stay green" or the expression of any gene that delays senescence would be advantageous. For example, a non-yellowing mutant has been identified in Festuca pratensis (Davies 1990). Expression of this gene as well as others may prevent premature breakdown of chlorophyll and thus maintain canopy function.

1.6 Nutrient Utilization The ability to utilize available nutrients and minerals may be a limiting factor in growth of many plants. It is proposed that it would be possible to alter nutrient uptake, tolerate pH extremes, mobilization through the plant, storage pools, and availability for meta- PF 56134 Sbolic activities by the introduction of novel genes. These modifications would allow a O plant to more efficiently utilize available nutrients. It is contemplated that an increase in N the activity of, for example, an enzyme that is normally present in the plant and involved in nutrient utilization would increase the availability of a nutrient. An example of S 5 such an enzyme would be phytase. It is also contemplated that expression of a novel gene may make a nutrient source available that was previously not accessible, an enzyme that releases a component of nutrient value from a more complex molecule, perhaps a macromolecule.

O 10 1.7. Non-Protein-Expressing Sequences _1.7.1 RNA-Expressing N DNA may be introduced into plants for the purpose of expressing RNA transcripts that function to affect plant phenotype yet are not translated into protein. Two examples are Santisense RNA and RNA with ribozyme activity. Both may serve possible functions in reducing or eliminating expression of native or introduced plant genes.

Genes may be constructed or isolated, which when transcribed, produce antisense RNA or double-stranded RNA that is complementary to all or part(s) of a targeted messenger RNA(s). The antisense RNA reduces production of the polypeptide product of the messenger RNA. The polypeptide product may be any protein encoded by the plant genome. The aforementioned genes will be referred to as antisense genes. An antisense gene may thus be introduced into a plant by transformation methods to produce a novel transgenic plant with reduced expression of a selected protein of interest. For example, the protein may be an enzyme that catalyzes a reaction in the plant. Reduction of the enzyme activity may reduce or eliminate products of the reaction which include any enzymatically synthesized compound in the plant such as fatty acids, amino acids, carbohydrates, nucleic acids and the like. Alternatively, the protein may be a storage protein, such as a zein, or a structural protein, the decreased expression of which may lead to changes in seed amino acid composition or plant morphological changes respectively. The possibilities cited above are provided only by way of example and do not represent the full range of applications.

Expression of antisense-RNA or double-stranded RNA by one of the expression cassettes of the invention is especially preferred. Also expression of sense RNA can be employed for gene silencing (co-suppression). This RNA is preferably a nontranslatable RNA. Gene regulation by double-stranded RNA ("double-stranded RNA interference"; dsRNAi) is well known in the arte and described for various organism including plants Matzke 2000; Fire A et al 1998; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364).

Genes may also be constructed or isolated, which when transcribed produce RNA enzymes, or ribozymes, which can act as endoribonucleases and catalyze the cleavage of RNA molecules with selected sequences. The cleavage of selected messenger RNA's can result in the reduced production of their encoded polypeptide products.

These genes may be used to prepare novel transgenic plants which possess them.

The transgenic plants may possess reduced levels of polypeptides including but not limited to the polypeptides cited above that may be affected by antisense RNA.

I PF 56134 56 SIt is also possible that genes may be introduced to produce novel transgenic plants which have reduced expression of a native gene product by a mechanism of cosup- C-i pression. It has been demonstrated in tobacco, tomato, and petunia (Goring 1991; O Smith 1990; Napoli 1990; van der Krol 1990) that expression of the sense transcript of a native gene will reduce or eliminate expression of the native gene in a manner similar to that observed for antisense genes. The introduced gene may encode all or part of the targeted native protein but its translation may not be required for reduction of levels of that native protein.

ID 10 1.7.2 Non-RNA-Expressing For example, DNA elements including those of transposable elements such as Ds, Ac, Sor Mu, may be, inserted into a gene and cause mutations. These DNA elements may Sbe inserted in order to inactivate (or activate) a gene and thereby "tag" a particular trait.

SIn this instance the transposable element does not cause instability of the tagged mutation, because the utility of the element does not depend on its ability to move in the c genome. Once a desired trait is tagged, the introduced DNA sequence may be used to clone the corresponding gene, using the introduced DNA sequence as a PCR primer together with PCR gene cloning techniques (Shapiro, 1983; Dellaporta 1988).

Once identified, the entire gene(s) for the particular trait, including control or regulatory regions where desired may be isolated, cloned and manipulated as desired. The utility of DNA elements introduced into an organism for purposed of gene tagging is independent of the DNA sequence and does not depend on any biological activity of the DNA sequence, transcription into RNA or translation into protein. The sole function of the DNA element is to disrupt the DNA sequence of a gene.

It is contemplated that unexpressed DNA sequences, including novel synthetic sequences could be introduced into cells as proprietary "labels" of those cells and plants and seeds thereof. It would not be necessary for a label DNA element to disrupt the function of a gene endogenous to the host organism, as the sole function of this DNA would be to identify the origin of the organism. For example, one could introduce a unique DNA sequence into a plant and this DNA element would identify all cells, plants, and progeny of these cells as having arisen from that labeled source. It is proposed that inclusion of label DNAs would enable one to distinguish proprietary germplasm or germplasm derived from such, from unlabelled germplasm.

Another possible element which may be introduced is a matrix attachment region element (MAR), such as the chicken lysozyme A element (Stief 1989), which can be positioned around an expressible gene of interest to effect an increase in overall expression of the gene and diminish position dependant effects upon incorporation into the plant genome (Stief 1989; Phi-Van 1990).

Further nucleotide sequences of interest that may be contemplated for use within the scope of the present invention in operable linkage with the promoter sequences according to the invention are isolated nucleic acid molecules, DNA or RNA, comprising a plant nucleotide sequence according to the invention comprising an open reading frame that is preferentially expressed in a specific tissue, meristem-, root, green tissue (leaf and stem), panicle-, or pollen, or is expressed constitutively.

PF 56134 57 n 2. Marker Genes 0In order to improve the ability to identify transformants, one may desire to employ a c selectable or screenable marker gene as, or in addition to, the expressible gene of interest. "Marker genes" are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can 'select' for by chemical means, through the use of a selective agent a herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing, by 'screening' the R-locus trait, the green fluorescent protein (GFP)).

Of course, many examples of suitable marker genes are known to the art and can be employed in the practice of the invention.

SIncluded within the terms selectable or screenable marker genes are also genes which encode a "secretable marker" whose secretion can be detected as a means of identify- CN ing or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes which can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, by ELISA; small active enzymes detectable in extracellular solution alpha-amylase, beta-lactamase, phosphinothricin acetyltransferase); and proteins that are inserted or trapped in the cell wall proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene that encodes a protein that becomes sequestered in the cell wall, and which protein includes a unique epitope is considered to be particularly advantageous. Such a secreted antigen marker would ideally employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that would impart efficient expression and targeting across the plasma membrane, and would produce protein that is bound in the cell wall and yet accessible to antibodies. A normally secreted wall protein modified to include a unique epitope would satisfy all such requirements.

One example of a protein suitable for modification in this manner is extensin, or hydroxyproline rich glycoprotein (HPRG). For example, the maize HPRG (Steifel 1990) molecule is well characterized in terms of molecular biology, expression and protein structure. However, any one of a variety of ultilane and/or glycine-rich wall proteins (Keller 1989) could be modified by the addition of an antigenic site to create a screenable marker.

One exemplary embodiment of a secretable screenable marker concerns the use of a maize sequence encoding the wall protein HPRG, modified to include a 15 residue epitope from the pro-region of murine interleukin, however, virtually any detectable epitope may be employed in such embodiments, as selected from the extremely wide variety of antigen-antibody combinations known to those of skill in the art. The unique extracellular epitope can then be straightforwardly detected using antibody labeling in conjunction with chromogenic or fluorescent adjuncts.

PF 56134 58 SElements of the present disclosure may be exemplified in detail through the use of the O bar and/or GUS genes, and also through the use of various other markers. Of course, N in light of this disclosure, numerous other possible selectable and/or screenable marker genes will be apparent to those of skill in the art in addition to the one set forth herein below. Therefore, it will be understood that the following discussion is exemplary rather than exhaustive. In light of the techniques disclosed herein and the general recombinant techniques which are known in the art, the present invention renders possible the introduction of any gene, including marker genes, into a recipient cell to generate a transformed plant.

\C 2.1 Selectable Markers N Various selectable markers are known in the art suitable for plant transformation. Such markers may include but are not limited to: 2.1.1 Negative selection markers CN Negative selection markers confer a resistance to a biocidal compound such as a metabolic inhibitor 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics kanamycin, G 418, bleomycin or hygromycin) or herbicides phosphinothricin or glyphosate). Transformed plant material cells, tissues or plantlets), which express marker genes, are capable of developing in the presence of concentrations of a corresponding selection compound antibiotic or herbicide) which suppresses growth of an untransformed wild type tissue. Especially preferred negative selection markers are those which confer resistance to herbicides. Examples which may be mentioned are: Phosphinothricin acetyltransferases (PAT; also named Bialophos ®resistance; bar; de Block 1987; Vasil 1992, 1993; Weeks 1993; Becker 1994; Nehra 1994; Wan Lemaux 1994; EP 0 333 033; US 4,975,374). Preferred are the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes. PAT inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami 1986; Twell 1989) causing rapid accumulation of ammonia and cell death.

altered 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) conferring resistance to Glyphosate® (N-(phosphonomethyl)glycine) (Hinchee 1988; Shah 1986; Della-Cioppa 1987). Where a mutant EPSP synthase gene is employed, additional benefit may be realized through the incorporation of a suitable chloroplast transit peptide, CTP (EP-A1 0 218 571).

-Glyphosate® degrading enzymes (Glyphosate® oxidoreductase; gox), -Dalapon® inactivating dehalogenases (deh) -sulfonylurea- and/or imidazolinone-inactivating acetolactate synthases (ahas or ALS; for example mutated ahas/ALS variants with, for example, the S4, X112, XA17, and/or Hra mutation (EP-A1 154 204) SBromoxynil® degrading nitrilases (bxn; Stalker 1988) -Kanamycin- or. geneticin (G418) resistance genes (NPTII; NPT or neo; Potrykus 1985) coding for neomycin phosphotransferases (Fraley 1983; Nehra 1994) 2-Desoxyglucose-6-phosphate phosphatase (DOGR1-Gene product; WO 98/45456; EP 0 807 836) conferring resistance against 2-desoxyglucose (Randez- Gil 1995).

hygromycin phosphotransferase (HPT), which mediates resistance to hygromycin r PF 56134 59 (Vanden Elzen 1985).

altered dihydrofolate reductase (Eichholtz 1987) conferring resistance against rC methotrexat (Thillet 1988); mutated anthranilate synthase genes that confers resistance to 5-methyl tryptophan.

Additional negative selectable marker genes of bacterial origin that confer resistance to antibiotics include the aadA gene, which confers resistance to the antibiotic spectinomycin, gentamycin acetyl transferase, streptomycin phosphotransferase (SPT), ami- IN 10 noglycoside-3-adenyl transferase and the bleomycin resistance determinant (Hayford S1988; Jones 1987; Svab 1990; Hille 1986).

Especially preferred are negative selection markers that confer resistance against the I toxic effects imposed by D-amino acids like D-alanine and D-serine (WO 03/060133; Erikson 2004). Especially preferred as negative selection marker in this contest are the daol gene (EC: 1.4. 3.3 GenBank Acc.-No.: U60066) from the yeast Rhodotorula gracilis (Rhodosporidium toruloides) and the E. coli gene dsdA (D-serine dehydratase (D-serine deaminase) [EC: 4.3. 1.18; GenBank Acc.-No.: J01603).

Transformed plant material cells, embryos, tissues or plantlets) which express such marker genes are capable of developing in the presence of concentrations of a corresponding selection compound antibiotic or herbicide) which suppresses growth of an untransformed wild type tissue. The resulting plants can be bred and hybridized in the customary fashion. Two or more generations should be grown in order to ensure that the genomic integration is stable and hereditary. Corresponding methods are described (Jenes 1993; Potrykus 1991).

Furthermore, reporter genes can be employed to allow visual screening, which may or may not (depending on the type of reporter gene) require supplementation with a substrate as a selection compound.

Various time schemes can be employed for the various negative selection marker genes. In case of resistance genes against herbicides or D-amino acids) selection is preferably applied throughout callus induction phase for about 4 weeks and beyond at least 4 weeks into regeneration. Such a selection scheme can be applied for all selection regimes. It is furthermore possible (although not explicitly preferred) to remain the selection also throughout the entire regeneration scheme including rooting.

For example, with the phosphinotricin resistance gene (bar) as the selective marker, phosphinotricin at a concentration of from about 1 to 50 mg/I may be included in the medium. For example, with the daol gene as the selective marker, D-serine or Dalanine at a concentration of from about 3 to 100 mg/l may be included in the medium.

Typical concentrations for selection are 20 to 40 mg/l. For example, with the mutated ahas genes as the selective marker, PURSUIT" at a concentration of from about 3 to 100 mg/I may be included in the medium. Typical concentrations for selection are 20 to mg/l.

r PF 56134 t) 2.1.2 Positive selection marker O Furthermore, positive selection marker can be employed. Genes like isopentenyltransr ferase from Agrobacterium tumefaciens (strain:P022; Genbank Acc.-No.: AB025109) may as a key enzyme of the cytokinin biosynthesis facilitate regeneration of trans- S 5 formed plants by selection on cytokinin-free medium). Corresponding selection methods are described (Ebinuma 2000a,b). Additional positive selection markers, which confer a growth advantage to a transformed plant in comparison with a nontransformed one, are described in EP-A 0 601 092. Growth stimulation selection markers may include (but shall not be limited to) P-Glucuronidase (in combination with D 10 a cytokinin glucuronide), mannose-6-phosphate isomerase (in combination with mannose), UDP-galactose-4-epimerase (in combination with galactose), wherein Cmannose-6-phosphate isomerase in combination with mannose is especially preferred.

I2.1.3 Counter-selection marker Counter-selection markers are especially suitable to select organisms with defined deleted sequences comprising said marker (Koprek 1999). Examples for counter- selection marker comprise thymidin kinases cytosine deaminases (Gleave 1999; Perera 1993; Stougaard 1993), cytochrom P450 proteins (Koprek 1999), haloalkan dehalogenases (Naested 1999), iaaH gene products (Sundaresan 1995), cytosine deaminase codA (Schlaman Hooykaas 1997), tms2 gene products (Fedoroff Smith 1993), or a-naphthalene acetamide (NAM; Depicker 1988). Counter selection markers may be useful in the construction of transposon tagging lines. For example, by marking an autonomous transposable element such as Ac, Master Mu, or En/Spn with a counter selection marker, one could select for transformants in which the autonomous element is not stably integrated into the genome. This would be desirable, for example, when transient expression of the autonomous element is desired to activate in trans the transposition of a defective transposable element, such as Ds, but stable integration of the autonomous element is not desired. The presence of the autonomous element may not be desired in order to stabilize the defective element, prevent it from further transposing. However, it is proposed that if stable integration of an autonomous transposable element is desired in a plant the presence of a negative selectable marker may make it possible to eliminate the autonomous element during the breeding process.

2.2. Screenable Markers Screenable markers that may be employed include, but are not limited to, a betaglucuronidase (GUS) or uidA gene which encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta 1988); a beta-lactamase gene (Sutcliffe 1978), which encodes an enzyme for which various chromogenic substrates are known PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky 1983) which encodes a catechol dioxygenase that can convert chromogenic catechols; an a-amylase gene (Ikuta 1990); a tyrosinase gene (Katz 1983) which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to form the easily detectable compound melanin; P-galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a luciferase (lux) gene (Ow 1986), which allows for bioluminescence detection; or even an aequorin gene (Prasher 1985), which may be employed in PF 56134 61 Scalcium-sensitive bioluminescence detection, or a green fluorescent protein gene O (Niedz 1995).

SGenes from the maize R gene complex are contemplated to be particularly useful as screenable markers. The R gene complex in maize encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue. A gene from the R gene complex was applied to maize transformation, because the expression of this gene in transformed cells does not harm the cells. Thus, an R gene introduced into such cells will cause the expression of a red pigment and, if stably incorporated, can be O 10 visually scored as a red sector. If a maize line is dominant for genes encoding the enzymatic intermediates in the anthocyanin biosynthetic pathway (C2, Al, A2, Bzl and Bz2), but carries a recessive allele at the R locus, transformation of any cell from that line with R will result in red pigment formation. Exemplary lines include Wisconsin 22 Swhich contains the rg-Stadler allele and TR 12, a K55 derivative which is r-g, b, P1.

Alternatively any genotype of maize can be utilized if the Cl and R alleles are introduced together.

It is further proposed that R gene regulatory regions may be employed in chimeric constructs in order to provide mechanisms for controlling the expression of chimeric genes.

More diversity of phenotypic expression is known at the R locus than at any other locus (Coe 1988). It is contemplated that regulatory regions obtained from regions 5' to the structural R gene would be valuable in directing the expression of genes, insect resistance, drought resistance, herbicide tolerance or other protein coding regions. For the purposes of the present invention, it is believed that any of the various R gene family members may be successfully employed P, S, Lc, etc.). However, the most preferred will generally be Sn (particularly Sn:bol3). Sn is a dominant member of the R gene complex and is functionally similar to the R and B loci in that Sn controls the tissue specific deposition of anthocyanin pigments in certain seedling and plant cells, therefore, its phenotype is similar to R.

A further screenable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene. The presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It is also envisioned that this system may be developed for populational screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening. Where use of a screenable marker gene such as lux or GFP is desired, benefit may be realized by creating a gene fusion between the screenable marker gene and a selectable marker gene, for example, a GFP-NPTII gene fusion.

This could allow, for example, selection of transformed cells followed by screening of transgenic plants or seeds.

3. Exemplary DNA Molecules The invention provides an isolated nucleic acid molecule, DNA or RNA, comprising a plant nucleotide sequence comprising an open reading frame that is preferentially expressed in a specific plant tissue, in seeds, roots, green tissue (leaf and stem), panicles or pollen, or is expressed constitutively, or a promoter thereof.

PF 56134 62 SThese promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, spatially-regulated, chemically regulated, stressri responsive, tissue-specific, viral and synthetic promoters. Promoter sequences are O known to be strong or weak. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression.

An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus. A bacterial promoter such as the Pta, promoter can be induced to varying levels of gene expression depending on the level of isothiopropylgalactoside ID 10 added to the transformed bacterial cells. An isolated promoter sequence that is a strong promoter for heterologous nucleic acid is advantageous because it provides for ri a sufficient level of gene expression to allow for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.

In Within a plant promoter region there are several domains that are necessary for full function of the promoter. The first of these domains lies immediately upstream of the structural gene and forms the "core promoter region" containing consensus sequences, normally 70 base pairs immediately upstream of the gene. The core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence that defines the transcription start point for the structural gene.

The presence of the core promoter region defines a sequence as being a promoter: if the region is absent, the promoter is non-functional. Furthermore, the core promoter region is insufficient to provide full promoter activity. A series of regulatory sequences upstream of the core constitute the remainder of the promoter. The regulatory sequences determine expression level, the spatial and temporal pattern of expression and, for an important subset of promoters, expression under inductive conditions (regulation by external factors such as light, temperature, chemicals, hormones).

Regulated expression of the chimeric transacting viral replication protein can be further regulated by other genetic strategies. For example, Cre-mediated gene activation as described by Odell et al. 1990. Thus, a DNA fragment containing 3' regulatory sequence bound by lox sites between the promoter and the replication protein coding sequence that blocks the expression of a chimeric replication gene from the promoter can be removed by Cre-mediated excision and result in the expression of the transacting replication gene. In this case, the chimeric Cre gene, the chimeric trans-acting replication gene, or both can be under the control of tissue- and developmental-specific or inducible promoters. An alternate genetic strategy is the use of tRNA suppressor gene. For example, the regulated expression of a tRNA suppressor gene can conditionally control expression of a trans-acting replication protein coding sequence containing an appropriate termination codon as described by Ulmasov et al. 1997. Again, either the chimeric tRNA suppressor gene, the chimeric transacting replication gene, or both can be under the control of tissue- and developmental-specific or inducible promoters.

PF 56134 63 V) Frequently it is desirable to have continuous or inducible expression of a DNA sequence throughout the cells of an organism in a tissue-independent manner. For ex- C ample, increased resistance of a plant t6 infection by soil- and airborne-pathogens O might be accomplished by genetic manipulation of the plant's genome to comprise a continuous promoter operably linked to a heterologous pathogen-resistance gene such that pathogen-resistance proteins are continuously expressed throughout the plant's tissues.

Alternatively, it might be desirable to inhibit expression of a native DNA sequence I0 10 within the seeds of a plant to achieve a desired phenotype. In this case, such inhibition might be accomplished with transformation of the plant to comprise a promoter opera- CN bly linked to an antisense nucleotide sequence, such that meristem-preferential or meristem-specific expression of the antisense sequence produces an RNA transcript that Iinterferes with translation of the mRNA of the native DNA sequence.

STo define a minimal promoter region, a DNA segment representing the promoter region is removed from the 5' region of the gene of interest and operably linked to the coding sequence of a marker (reporter) gene by recombinant DNA techniques well known to the art. The reporter gene is operably linked downstream of the promoter, so that transcripts initiating at the promoter proceed through the reporter gene. Reporter genes generally encode proteins which are easily measured, including, but not limited to, chloramphenicol acetyl transferase (CAT), beta-glucuronidase (GUS), green fluorescent protein (GFP), beta-galactosidase (beta-GAL), and luciferase.

The construct containing the reporter gene under the control of the promoter is then introduced into an appropriate cell type by transfection techniques well known to the art. To assay for the reporter protein, cell lysates are prepared and appropriate assays, which are well known in the art, for the reporter protein are performed. For example, if CAT were the reporter gene of choice, the lysates from cells transfected with constructs containing CAT under the control of a promoter under study are mixed with isotopically labeled chloramphenicol and acetyl-coenzyme A (acetyl-CoA). The CAT enzyme transfers the acetyl group from acetyl-CoA to the 2- or 3-position of chloramphenicol. The reaction is monitored by thin-layer chromatography, which separates acetylated chloramphenicol from unreacted material. The reaction products are then visualized by autoradiography.

The level of enzyme activity corresponds to the amount of enzyme that was made, which in turn reveals the level of expression from the promoter of interest. This level of expression can be compared to other promoters to determine the relative strength of the promoter under study. In order to be sure that the level of expression is determined by the promoter, rather than by the stability of the mRNA, the level of the reporter mRNA can be measured directly, such as by Northern blot analysis.

Once activity is detected, mutational and/or deletional analyses may be employed to determine the minimal region and/or sequences required to initiate transcription. Thus, sequences can be deleted at the 5' end of the promoter region and/or at the 3' end of PF 56134 64 t the promoter region, and nucleotide substitutions introduced. These constructs are 8 then introduced to cells and their activity determined.

0 In one embodiment, the promoter may be a gamma zein promoter, an oleosin ole16 S 5 promoter, a globulins promoter, an actin I promoter, an actin cl promoter, a sucrose synthetase promoter, an INOPS promoter, an EXM5 promoter, a globulin2 promoter, a b-32, ADPG-pyrophosphorylase promoter, an Ltpl promoter, an Ltp2 promoter, an oleosin ole17 promoter, an oleosin ole18 promoter, an actin 2 promoter, a pollenspecific protein promoter, a pollen-specific pectate lyase promoter, an anther-specific O 10 protein promoter, an anther-specific gene RTS2 promoter, a pollen-specific gene promoter, a tapeturn-specific gene promoter, tapeturn-specific gene RAB24 promoter, a anthranilate synthase alpha subunit promoter, an alpha zein promoter, an anthranilate synthase beta subunit promoter, a dihydrodipicolinate synthase promoter, a Thil pro- I moter, an alcohol dehydrogenase promoter, a cab binding protein promoter, an H3C4 promoter, a RUBISCO SS starch branching enzyme promoter, an ACCase promoter, an actin3 promoter, an actin7 promoter, a regulatory protein GF14-12 promoter, a ribosomal protein L9 promoter, a cellulose biosynthetic enzyme promoter, an S-adenosyl- L-homocysteine hydrolase promoter, a superoxide dismutase promoter, a C-kinase receptor promoter, a phosphoglycerate mutase promoter, a root-specific RCc3 mRNA promoter, a glucose-6 phosphate isomerase promoter, a pyrophosphate-fructose 6phosphatelphosphotransferase promoter, an ubiquitin promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDa photosystem 11 promoter, an oxygen evolving protein promoter, a 69 kDa vacuolar ATPase subunit promoter, a metallothionein-like protein promoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, an ABA- and ripening-inducible-like protein promoter, a phenylalanine ammonia lyase promoter, an adenosine triphosphatase S-adenosyl-L-homocysteine hydrolase promoter, an atubulin promoter, a cab promoter, a PEPCase promoter, an R gene promoter, a lectin promoter, a light harvesting complex promoter, a heat shock protein promoter, a chalcone synthase promoter, a zein promoter, a globulin-1 promoter, an ABA promoter, an auxin-binding protein promoter, a UDP glucose flavonoid glycosyl-transferase gene promoter, an NTI promoter, an actin promoter, an opaque 2 promoter, a b70 promoter, an oleosin promoter, a CaMV 35S promoter, a CaMV 34S promoter, a CaMV 19S promoter, a histone promoter, a turgor-inducible promoter, a pea small subunit RuBP carboxylase promoter, a Ti plasmid mannopine synthase promoter, Ti plasmid nopaline synthase promoter, a petunia chalcone isomerase promoter, a bean glycine rich protein I promoter, a CaMV 35S transcript promoter, a potato patatin promoter, or a S-E9 small subunit RuBP carboxylase promoter.

4. Transformed (Transgenic) Plants of the Invention and Methods of Preparation Plant species may be transformed with the DNA construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art.

Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector of the present invention. The term "organogenesis," as used herein, means a process by which shoots and roots are de- PF 56134 t veloped sequentially from meristematic centers; the term "embryogenesis," as used 8 herein, means a process by which shoots and roots develop together in a concerted c( fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best d 5 suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue apical meristems, axillary buds, and root meristems), and induced meristem tissue cotyledon meristem and ultilane meristem).

N 10 Plants of the present invention may take a variety of forms. The plants may be chimeras of transformed cells and non-transformed cells; the plants may be clonal transfor- Smants all cells transformed to contain the expression cassette); the plants may comprise grafts of transformed and untransformed tissues a transformed root I stock grafted to an untransformed scion in citrus species). The transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, first generation (or T1) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques. A dominant selectable marker (such as npt II) can be associated with the expression cassette to assist in breeding.

Thus, the present invention provides a transformed (transgenic) plant cell, in planta or ex planta, including a transformed plastid or other organelle, nucleus, mitochondria or chloroplast. The present invention may be used for transformation of any plant species, including, but not limited to, cells from the plant species specified above in the DEFINITION section. Preferably, transgenic plants of the present invention are crop plants and in particular cereals (for example, corn, alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), and even more preferably corn, rice and soybean. Other embodiments of the invention are related to cells, cell cultures, tissues, parts (such as plants organs, leaves, roots, etc.) and propagation material (such as seeds) of such plants.

The transgenic expression cassette of the invention may not only be comprised in plants or plant cells but may advantageously also be containing in other organisms such for example bacteria. Thus, another embodiment of the invention relates to transgenic cells or non-human, transgenic organisms comprising an expression cassette of the invention. Preferred are prokaryotic and eukaryotic organism. Both microorganism and higher organisms are comprised. Preferred microorganism are bacteria, yeast, algae, and fungi. Preferred bacteria are those of the genus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus or Cyanobacterim such as for example Synechocystis and other bacteria described in Brock Biology of Microorganisms Eighth Edition (pages A-8, A-9, A10 and Al 1).

Especially preferred are microorganisms capable to infect plants and to transfer DNA into their genome, especially bacteria of the genus Agrobacterium, preferably Agrobacterium tumefaciens and rhizogenes. Preferred yeasts are Candida, Saccharomyces, PF 56134 66 Hansenula and Pichia. Preferred Fungi are Aspergillus, Trichoderma, Ashbya, Neuro- O spora, Fusarium, and Beauveria. Most preferred are plant organisms as defined above.

0 Transformation of plants can be undertaken with a single DNA molecule or multiple d 5 DNA molecules co-transformation), and both these techniques are suitable for use with the expression cassettes of the present invention. Numerous transformation vectors are available for plant transformation, and the expression cassettes of this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transfor- I 10 mation.

CA variety of techniques are available and known to those skilled in the art for introduction of constructs into a plant cell host. These techniques generally include transformation with DNA employing A. tumefaciens or A. rhizogenes as the transforming agent, 0 15 liposomes, PEG precipitation, electroporation, DNA injection, direct DNA uptake, microprojectile bombardment, particle acceleration, and the like (See, for example, EP 295959 and EP 138341) (see below). However, cells other than plant cells may be transformed with the expression cassettes of the invention. The general descriptions of plant expression vectors and reporter genes, and Agrobacterium and Agrobacteriummediated gene transfer, can be found in Gruber et al. (1993).

Expression vectors containing genomic or synthetic fragments can be introduced into protoplasts or into intact tissues or isolated cells. Preferably expression vectors are introduced into intact tissue. General methods of culturing plant tissues are provided for example by Maki et al., (1993); and by Phillips et al. (1988). Preferably, expression vectors are introduced into maize or other plant tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation and the like. More preferably expression vectors are introduced into plant tissues using the microprojectile media delivery with the biolistic device. See, for example, Tomes et al.

(1995). The vectors of the invention can not only be used for expression of structural genes but may also be used in exon-trap cloning, or promoter trap procedures to detect differential gene expression in varieties of tissues (Lindsey 1993; Auch Reth 1990).

It is particularly preferred to use the binary type vectors of Ti and Ri plasmids of Agrobacterium spp. Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti 1985: Byrne 1987; Sukhapinda 1987; Lorz 1985; Potrykus, 1985; Park 1985: Hiei 1994). The use of T-DNA to transform plant cells has received extensive study and is amply described (EP 120516; Hoekema, 1985; Knauf, 1983; and An 1985). For introduction into plants, the chimeric genes of the invention can be inserted into binary vectors as described in the examples.

Other transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs (see EP 295959), techniques of electroporation (Fromm 1986) or high velocity ballistic bombardment with metal particles coated with the nucleic acid constructs (Kline 1987, and US 4,945,050). Once transformed, the cells can be regenerated by those skilled in the art. Of particular relevance are the re-

I

PF 56134 67 0 cently described methods to transform foreign genes into commercially important Scrops, such as rapeseed (De Block 1989), sunflower (Everett 1987), soybean (McCabe cN 1988; Hinchee 1988; Chee 1989; Christou 1989; EP 301749), rice (Hiei 1994), and 0 corn (Gordon-Kamm 1990; Fromm 1990).

Those skilled in the art will appreciate that the choice of method might depend on the type of plant, monocotyledonous or dicotyledonous, targeted for transformation.

Suitable methods of transforming plant cells include, but are not limited to, microinjection (Crossway 1986), electroporation (Riggs 1986), Agrobacterium-mediated transfor- O 10 mation (Hinchee 1988), direct gene transfer (Paszkowski 1984), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wis. And BioRad, C1 Hercules, Calif. (see, for example, US 4,945,050; and McCabe 1988). Also see, Weissinger 1988; Sanford 1987 (onion); Christou 1988 (soybean); McCabe 1988 (soy- Ibean); Datta 1990 (rice); Klein 1988 (maize); Klein 1988 (maize); Klein 1988 (maize); Fromm 1990 (maize); and Gordon-Kamm 1990 (maize); Svab 1990 (tobacco chloro- Splast); Koziel 1993 (maize); Shimamoto 1989 (rice); Christou 1991 (rice); European Patent Application EP 0 332 581 (orchardgrass and other Pooideae); Vasil 1993 (wheat); Weeks 1993 (wheat).

In another embodiment, a nucleotide sequence of the present invention is directly transformed into the plastid genome. Plastid transformation technology is extensively described in US 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride et al., 1994. The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, using biolistics or protoplast transformation calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate orthologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab 1990; Staub 1992). This resulted in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub 1993). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3N-adenyltransferase (Svab 1993). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the invention. Typically, approximately 15-20 cell division cycles following transformation are required to reach a homoplastidic state. Plastid expression, in which genes are inserted by orthologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein. In a preferred embodiment, a nucleotide sequence of the present invention is inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for PF 56134 68 t plastid genomes containing a nucleotide sequence of the present invention are ob- O tained, and are preferentially capable of high expression of the nucleotide sequence.

0 Agrobacterium tumefaciens cells containing a vector comprising an expression cassette of the present invention, wherein the vector comprises a Ti plasmid, are useful in methods of making transformed plants. Plant cells are infected with an Agrobacterium tumefaciens as described above to produce a transformed plant cell, and then a plant is regenerated from the transformed plant cell. Numerous Agrobacterium vector systems useful in carrying out the present invention are known.

I\O Various Agrobacterium strains can be employed, preferably disarmed Agrobacterium Stumefaciens or rhizogenes strains. In a preferred embodiment, Agrobacterium strains for use in the practice of the invention include octopine strains, LBA4404 or ag- Iropine strains, EHA101 or EHA105. Suitable strains of A. tumefaciens for DNA 0 15 transfer are for example EHA101[pEHA101] (Hood 1986), EHA105[pEHA105] (Li 1992), LBA4404[pAL4404] (Hoekema 1983), C58C1[pMP90] (Koncz Schell 1986), and C58C1[pGV2260] (Deblaere 1985). Other suitable strains are Agrobacterium tumefaciens C58, a nopaline strain. Other suitable strains are A. tumefaciens C58C1 (Van Larebeke 1974), A136 (Watson 1975) or LBA4011 (Klapwijk 1980). In another preferred embodiment the soil-borne bacterium is a disarmed variant of Agrobacterium rhizogenes strain K599 (NCPPB 2659). Preferably, these strains are comprising a disarmed plasmid variant of a Ti- or Ri-plasmid providing the functions required for T-DNA transfer into plant cells the vir genes). In a preferred embodiment, the Agrobacterium strain used to transform the plant tissue pre-cultured with the plant phenolic compound contains a L,L-succinamopine type Ti-plasmid, preferably disarmed, such as pEHA101. In another preferred embodiment, the Agrobacterium strain used to transform the plant tissue pre-cultured with the plant phenolic compound contains an octopine-type Ti-plasmid, preferably disarmed, such as pAL4404. Generally, when using octopine-type Ti-plasmids or helper plasmids, it is preferred that the virF gene be deleted or inactivated (Jarschow 1991).

The method of the invention can also be used in combination with particular Agrobacterium strains, to further increase the transformation efficiency, such as Agrobacterium strains wherein the vir gene expression and/or induction thereof is altered due to the presence of mutant or chimeric virA or virG genes Hansen 1994; Chen and Winans 1991; Scheeren-Groot, 1994). Preferred are further combinations of Agrobacterium tumefaciens strain LBA4404 (Hiei 1994) with super-virulent plasmids. These are preferably pTOK246-based vectors (Ishida 1996).

A binary vector or any other vector can be modified by common DNA recombination techniques, multiplied in E. coli, and introduced into Agrobacterium by electroporation or other transformation techniques (Mozo Hooykaas 1991).

Agrobacterium is grown and used in a manner similar to that described in Ishida (1996). The vector comprising Agrobacterium strain may, for example, be grown for 3 days on YP medium (5 g/l yeast extract, 10 g/l peptone, 5 g/l NaCI, 15 g/l agar, pH 6.8) supplemented with the appropriate antibiotic 50 mg/I spectinomycin). Bacteria are PF 56134 69 0 collected with a loop from the solid medium and resuspended. In a preferred embodiment of the invention, Agrobacterium cultures are started by use of aliquots frozen at CN 80 0

C.

The transformation of the target tissue an immature embryo) by Agrobacterium may be carried out by merely contacting the target tissue with Agrobacterium. The concentration of Agrobacterium used for infection and co-cultivation may need to be varied. For example, a cell suspension of the Agrobacterium having a population density of approximately from 10' 101, preferably 106 to 1010, more preferably about 108 cells I 10 or cfu ml is prepared and the target tissue is immersed in this suspension for about 3 Sto 10 minutes. The resulting target tissue is then cultured on a solid medium for several Cl days together with the Agrobacterium.

I Preferably, the bacterium is employed in concentration of 106 to 1010 cfu/ml. In a pre- O 15 ferred embodiment for the co-cultivation step about 1 to 10 pl of a suspension of the soil-borne bacterium Agrobacteria) in the co-cultivation medium are directly applied to each target tissue explant and air-dried. This is saving labor and time and is reducing unintended Agrobacterium-mediated damage by excess Agrobacterium usage.

For Agrobacterium treatment, the bacteria are resuspended in a plant compatible cocultivation medium. Supplementation of the co-culture medium with antioxidants silver nitrate), phenol-absorbing compounds (like polyvinylpyrrolidone, Perl 1996) or thiol compounds dithiothreitol, L-cysteine, Olhoft 2001) which can decrease tissue necrosis due to plant defence responses (like phenolic oxidation) may further improve the efficiency of Agrobacterium-mediated transformation. In another preferred embodiment, the co-cultivation medium of comprises least one thiol compound, preferably selected from the group consisting of sodium thiolsulfate, dithiotrietol (DTT) and cysteine. Preferably the concentration is between about 1 mM and 10mM of L- Cysteine, 0.1 mM to 5 mM DTT, and/or 0.1 mM to 5 mM sodium thiolsulfate. Preferably, the medium employed during co-cultivation comprises from about 1 pM to about pM of silver nitrate and from about 50 mg/L to about 1,000 mg/L of L-Cystein. This results in a highly reduced vulnerability of the target tissue against Agrobacteriummediated damage (such as induced necrosis) and highly improves overall transformation efficiency.

Various vector systems can be used in combination with Agrobacteria. Preferred are binary vector systems. Common binary vectors are based on "broad host range"plasmids like pRK252 (Bevan 1984) or pTJS75 (Watson 1985) derived from the P-type plasmid RK2. Most of these vectors are derivatives of pBIN19 (Bevan 1984). Various binary vectors are known, some of which are commercially available such as, for example, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA). Additional vectors were improved with regard to size and handling pPZP; Hajdukiewicz 1994). Improved vector systems are described also in WO 02/00900.

Methods using either a form of direct gene transfer or Agrobacterium-mediated transfer usually, but not necessarily, are undertaken with a selectable marker which may pro- PF 56134 l vide resistance to an antibiotic kanamycin, hygromycin or methotrexate) or a herbicide phosphinothricin). The choice of selectable marker for plant transformation N is not, however, critical to the invention.

For certain plant species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene O which confers resistance to kanamycin and related antibiotics (Messing Vierra, 1982; Bevan 1983), the bar gene which confers resistance to the herbicide phosphinothricin (White 1990, Spencer 1990), the hph gene which confers resistance to the antibiotic O 10 hygromycin (Blochlinger Diggelmann), and the dhfr gene, which confers resistance to methotrexate (Bourouis 1983).

-i 5. Production and Characterization of Stably Transformed Plants STransgenic plant cells are then placed in an appropriate selective medium for selection of transgenic cells which are then grown to callus. Shoots are grown from callus and CN plantlets generated from the shoot by growing in rooting medium. The various constructs normally will be joined to a marker for selection in plant cells. Conveniently, the marker may be resistance to a biocide (particularly an antibiotic, such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide, or the like). The particular marker used will allow for selection of transformed cells as compared to cells lacking the DNA which has been introduced. Components of DNA constructs including transcription cassettes of this invention may be prepared from sequences which are native (endogenous) or foreign (exogenous) to the host. By "foreign" it is meant that the sequence is not found in the wild-type host into which the construct is introduced. Heterologous constructs will contain at least one region which is not native to the gene from which the transcription-initiation-region is derived.

To confirm the presence of the transgenes in transgenic cells and plants, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, in situ hybridization and nucleic acid-based amplification methods such as PCR or RT- PCR; "biochemical" assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as seed assays; and also, by analyzing the phenotype of the whole regenerated plant, for disease or pest resistance.

DNA may be isolated from cell lines or any plant parts to determine the presence of the preselected nucleic acid segment through the use of techniques well known to those skilled in the art. Note that intact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell.

The presence of nucleic acid elements introduced through the methods of this invention may be determined by polymerase chain reaction (PCR). Using this technique discreet fragments of nucleic acid are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a preselected nucleic acid segment is present in a stable transformant, but does not prove integration of the introduced preselected nucleic acid segment into the host cell genome. In addition, it is not possir PF 56134 71 Sble using PCR techniques to determine whether transformants have exogenous genes introduced into different sites in the, genome, whether transformants are of inder pendent origin. It is contemplated that using PCR techniques it would be possible to U clone fragments of the host genomic DNA adjacent to an introduced preselected DNA S 5 segment.

Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization.

Using this technique specific DNA sequences that were introduced into the host ge- O 10 nome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that C transformant. In addition it is possible through Southern hybridization to demonstrate the presence of introduced preselected DNA segments in high molecular weight DNA, Si.e., confirm that the introduced preselected, DNA segment has been integrated into the host cell genome. The technique of Southern hybridization provides information that is Sobtained using PCR, the presence of a preselected DNA segment, but also demonstrates integration into the genome and characterizes each individual transformant.

It is contemplated that using the techniques of dot or slot blot hybridization which are modifications of Southern hybridization techniques one could obtain the same information that is derived from PCR, the presence of a preselected DNA segment.

Both PCR and Southern hybridization techniques can be used to demonstrate transmission of a preselected DNA segment to progeny. In most instances the characteristic Southern hybridization pattern for a given transformant will segregate in progeny as one or more Mendelian genes (Spencer 1992); Laursen 1994) indicating stable inheritance of the gene. The non-chimeric nature of the callus and the parental transformants

(R

0 was suggested by germline transmission and the identical Southern blot hybridization patterns and intensities of the transforming DNA in callus, R 0 plants and R 1 progeny that segregated for the transformed gene.

Whereas DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA may only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues. PCR techniques may also be used for detection and quantitation of RNA produced from introduced preselected DNA segments. In this application of PCR it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. In most instances PCR techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species.

PF 56134 72 SWhile Southern blotting and PCR may be used to detect the preselected DNA segment in question, they do not provide information as to whether the preselected DNA seg- C ment is being expressed. Expression may be evaluated by specifically identifying the U protein products of the introduced preselected DNA segments or evaluating the phenotypic changes brought about by their expression.

Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physicalchemical or structural properties allow the proteins to be separated and identified by ID 10 electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelec- _tric focusing, or by chromatographic techniques such as ion exchange or gel exclusion r chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay.

SCombinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products that N have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.

Assay procedures may also be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed.

Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Morphological changes may include greater stature or thicker stalks. Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays.

6. Uses of Transgenic Plants Once an expression cassette of the invention has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques. Particularly preferred plants of the invention include the agronomically important crops listed above. The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction and can thus be maintained and propagated in progeny plants. The present invention also relates to a transgenic plant cell, tissue, organ, seed or plant part obtained from the transgenic plant. Also included within the invention are transgenic descendants of the plant as well as transgenic plant cells, tissues, organs, seeds and plant parts obtained from the descendants.

I

PF 56134 73 SPreferably, the expression cassette in the transgenic plant is sexually transmitted. In Sone preferred embodiment, the coding sequence is sexually transmitted through a complete normal sexual cycle of the RO plant to the R1 generation. Additionally pre- O ferred, the expression cassette is expressed in the cells, tissues, seeds or plant of a transgenic plant in an amount that is different than the amount in the cells, tissues, seeds or plant of a plant which only differs in that the expression cassette is absent.

The transgenic plants produced herein are thus expected to be useful for a variety of commercial and research purposes. Transgenic plants can be created for use in tradi- ID 10 tional agriculture to possess traits beneficial to the grower agronomic traits such _as resistance to water deficit, pest resistance, herbicide resistance or increased yield), r, beneficial to the consumer of the grain harvested from the plant improved nutritive content in human food or animal feed; increased vitamin, amino acid, and antioxi- Sdant content; the production of antibodies (passive immunization) and nutriceuticals), or beneficial to the food processor improved processing traits). In such uses, the Splants are generally grown for the use of their grain in human or animal foods. Additionally, the use of root-specific promoters in transgenic plants can provide beneficial traits that are localized in the consumable (by animals and humans) roots of plants such as carrots, parsnips, and beets. However, other parts of the plants, including stalks, husks, vegetative parts, and the like, may also have utility, including use as part of animal silage or for ornamental purposes. Often, chemical constituents oils or starches) of maize and other crops are extracted for foods or industrial use and transgenic plants may be created which have enhanced or modified levels of such components.

Transgenic plants may also find use in the commercial manufacture of proteins or other molecules, where the molecule of interest is extracted or purified from plant parts, seeds, and the like. Cells or tissue from the plants may also be cultured, grown in vitro, or fermented to manufacture such molecules. The transgenic plants may also be used in commercial breeding programs, or may be crossed or bred to plants of related crop species. Improvements encoded by the expression cassette may be transferred, e.g., from maize cells to cells of other species, by protoplast fusion.

The transgenic plants may have many uses in research or breeding, including creation of new mutant plants through insertional mutagenesis, in order to identify beneficial mutants that might later be created by traditional mutation and selection. An example would be the introduction of a recombinant DNA sequence encoding a transposable element that may be used for generating genetic variation. The methods of the invention may also be used to create plants having unique "signature sequences" or other marker sequences which can be used to identify proprietary lines or varieties.

Thus, the transgenic plants and seeds according to the invention can be used in plant breeding which aims at the development of plants with improved properties conferred by the expression cassette, such as tolerance of drought, disease, or other stresses.

The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate descendant plants. Depending on the desired properties different r PF 56134 74 breeding measures are taken. The relevant techniques are well known in the art and 8 include but are not limited to hybridization, inbreeding, backcross breeding, multilane c breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines which for example increase the effectiveness of conventional 10 methods such as herbicide or pesticide treatment or allow to dispense with said methods due to their modified genetic properties. Alternatively new crops with improved Sstress tolerance can be obtained which, due to their optimized genetic "equipment", yield harvested product of better quality than products which were not able to tolerate I comparable adverse developmental conditions.

SEXAMPLES

Materials and General Methods Unless indicated otherwise, chemicals and reagents in the Examples were obtained from Sigma Chemical Company (St. Louis, MO), restriction endonucleases were from New England Biolabs (Beverly, MA) or Roche (Indianapolis, IN), oligonucleotides were synthesized by MWG Biotech Inc. (High Point, NC), and other modifying enzymes or kits regarding biochemicals and molecular biological assays were from Clontech (Palo Alto, CA), Pharmacia Biotech (Piscataway, NJ), Promega Corporation (Madison, WI), or Stratagene (La Jolla, CA). Materials for cell culture media were obtained from Gibco/BRL (Gaithersburg, MD) or DIFCO (Detroit, MI). The cloning steps carried out for the purposes of the present invention, such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, growing bacteria, multiplying phages and sequence analysis of recombinant DNA, are carried out as described by Sambrook (1989). The sequencing of recombinant DNA molecules is carried out using ABI laser fluorescence DNA sequencer following the method of Sanger (Sanger 1977).

For generating transgenic Arabidopsis plants Agrobacterium tumefaciens (strain C58C1[pMP90]) is transformed with the various promoter::GUS vector constructs (see below). Resulting Agrobacterium strains are subsequently employed to obtain transgenic plants. For this purpose a isolated transformed Agrobacterium colony is incubated in 4 ml culture (Medium: YEB medium with 50 pg/ml Kanamycin and 25 pg/ml Rifampicin) over night at 28°C. With this culture a 400 ml culture of the same medium is inoculated and incubated over night (28 220 rpm). The bacteria a precipitated by centrifugation (GSA-Rotor, 8.000 U/min, 20 min) and the pellet is resuspended in infiltration medium (1/2 MS-Medium; 0,5 g/l MES, pH 5,8; 50 g/l sucrose). The suspension is placed in a plant box (Duchefa) and 100 ml SILVET L-77 (Osi Special-ties Inc., Cat.

P030196) are added to a final concentration of 0.02%. The plant box with 8 to 12 Plants is placed into an exsiccator for 10 to 15 min. under vacuum with subsequent, spontaneous ventilation (expansion). This process is repeated 2-3 times. Thereafter all plants are transferred into pods with wet-soil and grown under long daytime conditions r PF 56134 n (16 h light; day temperature 22-24°C, night temperature 19 0 C; 65% rel. humidity).

Seeds are harvested after 6 weeks.

EXAMPLE 1: Growth conditions for plants for tissue-specific expression analysis To obtain 4 and 7 days old seedlings, about 400 seeds (Arabidopsis thaliana ecotype Columbia) are sterilized with a 80% ethanol:water solution for 2 minutes, treated with a sodium hypochlorite solution v/v) for 5 minutes, washed three times with distillated water and incubated at 4 0 C for 4 days to ensure a standardized germination.

Subsequently, seeds are incubated on Petri dishes with MS medium (Sigma M5519) supplemented with 1% sucrose, 0.5 g/l MES (Sigma M8652), 0.8% Difco-BactoAgar (Difco 0140-01), adjusted to pH 5.7. The seedlings are grown under 16 h light 8 h dark cyklus (Philips 58W/33 white light) at 22 0 C and harvested after 4 or 7 days, re- C spectively.

To obtain root tissue, 100 seeds are sterilized as described above, incubated at 4 0 C for

C

N 4 days, and transferred into 250ml flasks with MS medium (Sigma M5519) supplemented with additional 3% sucrose and 0.5 g/l MES (Sigma M8652), adjusted to pH 5.7 for further growing. The seedlings are grown at a 16 h light 8 h dark cycle (Philips 58W/33 white light) at 22°C and 120 rpm and harvested after 3 weeks. For all other plant organs employed, seeds are sown on standard soil (Type VM, Manna-Italia, Via S. Giacomo 42, 39050 San Giacomo/ Laives, Bolzano, Italien), incubated for 4 days at 4°C to ensure uniform germination, and subsequently grown under a 16 h light 8 darkness regime (OSRAM Lumi-lux Daylight 36W/12) at 22 0 C. Young rosette leaves are harvested at the 8-leaf stage (after about 3 weeks), mature rosette leaves are harvested after 8 weeks briefly before stem formation. Apices of out-shooting stems are harvested briefly after out-shooting. Stem, stem leaves, and flower buds are harvested in development stage 12 (Bowmann J Arabidopsis, Atlas of Morphology, Springer New York, 1995) prior to stamen development. Open flowers are harvested in development stage 14 immediately after stamen development. Wilting flowers are harvested in stage 15 to 16. Green and yellow shoots used for the analysis have a length of 10 to 13 mm.

EXAMPLE 2: Demonstration of expression profile To demonstrate and analyze the transcription regulating properties of a promoter of the useful to operably link the promoter or its fragments to a reporter gene, which can be employed to monitor its expression both qualitatively and quantitatively. Preferably bacterial IM-glucuronidase is used (Jefferson 1987). R-glucuronidase activity can be monitored in planta with chromogenic substrates such as 5-bromo-4-Chloro-3-indolyl-13-Dglucuronic acid during corresponding activity assays (Jefferson 1987). For determination of promoter activity and tissue specificity plant tissue is dissected, embedded, stained and analyzed as described Baumlein 1991).

For quantitative B-glucuronidase activity analysis MUG (methylumbelliferyl glucuronide) is used as a substrate, which is converted into MU (methylumbelliferone) and glucuronic acid. Under alkaline conditions this conversion can be quantitatively monitored fluorometrically (excitation at 365 nm, measurement at 455 nm; SpectroFluorimeter Thermo Life Sciences Fluoroscan) as described (Bustos 1989).

PF 56134 76 EXAMPLE 3: Cloning of the promoter fragments To isolate the promoter fragments described by SEQ I D NO: 1, 2, 3, 6, 7, 8, 11, 12, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, and 36, genomic DNA is isolated from Arabidopsis thaliana (ecotype Columbia) as described (Galbiati 2000). The isolated genomic DNA is employed as matrix DNA for a polymerase chain reaction (PCR) mediated amplification using the oligonucleotide primers and protocols indicated below (Table 3) Table 3: PCR conditions and oligonucleotide primers for amplification of the various transcrip- Restriction Seq ID No. Promoter Forward Primer Reverse Primer Ta enzymes UH438for UH438rev SEQ ID NO: 1 pSUH438 SEQ ID NO: 37 SEQ ID NO: 38 52 0 C Xhol/Ncol UH438for UH438rev SEQ ID NO: 2 pSUH438GB SEQ ID NO: 37 SEQ ID NO: 38 52 0 C Xhol/NcoI UH438vfor UH438vrev SEQ ID NO: 3 pSUH438v SEQ ID NO: 39 SEQ ID NO: 40 55*C BamHl/NcoI UH436for UH436rev SEQ ID NO: 6 pSUH436 SEQ ID NO: 41 SEQ ID NO: 42 51 *C BamHl/Ncol UH436for UH436rev SEQ ID NO: 7 pSUH436GB SEQ ID NO: 41 SEQ ID NO: 42 51 *C BamHI/NcoI UH436for UH436Srev SEQ ID NO: 8 pSUH436S SEQ ID NO: 41 SEQ ID NO: 43 51'C BamHlfNcol UH433for UH433rev SEQ ID NO: 11 pSUH433 SEQ ID NO: 44 SEQ ID NO: 45 54 0 C BamHI/Ncol UH433for UH433rev SEQ ID NO: 12 pSUH433GB SEQ ID NO: 44 SEQ ID NO: 45 54 0 C BamHI/Ncol SEQ ID NO: 15 pSUH415 SEQ ID NO: 46 SEQ ID NO: 47 56*C BamHl/Ncol UH415rev SEQ ID NO: 16 pSUH4I5GB SEQ ID NO: 46 SEQ ID NO: 47 56 0 C BamHI/NcoI UH415Lrev SEQ ID NO: 17 pSUH4I5L SEQ ID NO: 48 SEQ ID NO: 49 56'C Xhol/NcoI UH4l6for UH4l6rev SEQ ID NO: 20 pSUH4I6 SEQ ID NO: 50 SEQ ID NO: 51 54 0 C BamHI/NcoI UH4l6for UH4l6rev SEQ ID NO: 21 pSUH416GB SEQ ID NO: 50 SEQ ID NO: 51 54 0 C BamHI/NcoI UH4l7for UH4l7rev SEQ ID NO: 24 DSUH417 SEQ ID NO: 52 SEQ ID NO: 53 5700 BamHI/NcoI UH4l7for UH4l7rev SEQ ID NO: 25 pSUH417GB SEQ ID NO: 52 SEQ ID NO: 53 57 0 C BamHI/Ncol UH432for UH432rev SEQ ID NO: 28 pSUH432 SEQ ID NO: 54 SEQ ID NO: 55 52 0 C BamHI/Spel UH43lfor UH43lrev SEQ ID NO: 31 DSUH431 SEQ ID NO: 56 SEQ ID NO: 57 56*C Xhol/BamHI- UH43lfor UH43lrev SEQ ID NO: 32 pSUH431GB SEQ ID NO: 56 SEQ ID NO: 57 560C Xhol/BamHI UH4l3for UH4l3rev SEQ ID NO: 35 pSUH4I3 SEQ ID NO: 58 SEQ ID NO: 59 4900 Xhol/NcoI UH4l3for UH4l3rev SEQ ID NO: 36 pSUH413GB SEQ ID NO: 58 SEQ ID NO: 59 1490C Xhol/NcoI PF 56134 77 0V Amplification is carried out as follows:

O

C1 100 ng genomic DNA S1X PCR buffer 2,5 mM MgCl 2 200 pM each of dATP, dCTP, dGTP und dTTP pmol of each oligonucleotide primers Units Pfu DNA Polymerase (Stratagene) in a final volume of 50 pl IC SThe following temperature program is employed for the various amplifications (BIORAD Cl Thermocycler).

I 1. 95C for 5 min 2. Ta°C for 1 min, followed by 72"C for 5 min and 95°C for 30 sec. Repeated 25 times.

3. Ta°C for 1 min, followed by 72°C for 4. Storage at 4°C The resulting PCR-products are digested with the restriction endonucleases specified in the Table above (Table 3) and cloned into the vector pSUN0301 (SEQ ID NO: (pre-digested with the same enzymes) upstream and in operable linkage to the glucuronidase (GUS) gene. Following stable transformation of each of these constructs into Arabidopsis thaliana tissue specificity and expression profile was analyzed by a histochemical and quantitative GUS-assay, respectively.

EXAMPLE 4: Expression profile of the various promoter::GUS constructs in stably transformed A. thaliana plants 4.1 pSUH415, pSUH415L and pSUH415GB The promoter sequences derived from gene At2g01180 confer a strong expression in shoot apical meristems of seedlings, in vegetative shoot meristems of young plants as well in primordia of lateral roots. No expression is observed in leaves, stem, flowers, shoots and seeds.

4.2 pSUH416 and pSUH416GB The promoter sequences derived from gene At3g45560 confer a strong expression in shoot apical meristems of seedlings, in vegetative shoot meristems of young plants as well in marginal meristems. No expression is observed in seedlings, leaves, stem, flowers, shoots and seeds.

4.3 pSUH413, and pSUH413GB The promoter sequences demonstrate an extraordinary strong expression in hypocotyls and in the apical meristem region of seedlings. In adult plants expression is observed in meristematic tissues of nodes, in interface between petioles and siliques and in branching points of petioles of inflorescences. Weak side activities are observed in vasculature tissue of leaves. No expression is observed in leaves, stem, flowers, shoots and seeds.

PF 56134 78 4.4 pSUH438, pSUH438v and pSUH438GB The promoter sequences derived from gene At2g02180 confer a strong expression in Cl shoot apical meristems of seedlings, in vegetative shoot meristems of young plants as O well in root tips. There are side activities in main vein of hypocotyl and roots of seedlings. No expression is observed in leaves, stem, flowers, shoots and seeds.

pSUH431 and pSUH431GB The promoter sequences derived from gene At4g11490 drive expression in shoot apical meristems in seedlings. In adult plants expression is observed in shoot meristems D 10 of nodes and in branching points of petioles of inflorescences. There are side activities in anthers. No expression is observed in leaves, stem, flowers, shoots and seeds.

4.6 pSUH417 and pSUH417GB IThe promoter sequences derived from gene At4g00580 drive expression in shoot apical meristems of seedlings. In adult plants expression is observed in vegetative shoot Smeristems and in interface between petioles and siliques. No expression is observed in leaves, stem, flowers, shoots and seeds.

4.7 pSUH432 The promoter sequences derived from gene At1g54480 drive expression in shoot apical meristems. No expression is observed in leaves, stem, flowers, shoots and seeds.

4.8 pSUH433 and pSUH433GB The promoter sequences derived from gene At2g26970 confer strong expression in shoot apex of seedlings. In adult plants expression is observed in shoot apex shoot meristems of nodes and in interface between petioles and siliques. There are side activities in root tips. No expression is observed in leaves, stem, flowers, shoots and seeds.

4.9 pSUH436, pSUH436S and pSUH436GB The promoter sequences derived from gene At5g54510 demonstrate strong expression in shoot apex of seedlings. In adult plants expression is observed in shoot apex shoot meristems of nodes and in interface between petioles and siliques. There are side activities in root tips. No expression is observed in leaves, stem, flowers, shoots and seeds.

EXAMPLE 5 Vector Construction for Overexpression and Gene "Knockout" Experiments 5.1 Overexpression Vectors used for expression of full-length "candidate genes" of interest in plants (overexpression) are designed to overexpress the protein of interest and are of two general types, biolistic and binary, depending on the plant transformation method to be used.

For biolistic transformation (biolistic vectors), the requirements are as follows: PF 56134 79 1. a backbone with a bacterial selectable marker (typically, an antibiotic resistance O gene) and origin of replication functional in Escherichia coli coli ColE1), Ci and 2. a plant-specific portion consisting of: a. a gene expression cassette consisting of a promoter (eg. ZmUBlint MOD), the gene of interest (typically, a full-length cDNA) and a transcriptional terminator Agrobacterium tumefaciens nos terminator); b. a plant selectable marker cassette, consisting of a suitable promoter, selectable I marker gene D-amino acid oxidase; daol) and transcriptional terminator C 10 (eg. nos terminator).

Vectors designed for transformation by Agrobacterium tumefaciens tumefaciens; In binary vectors) consist of: S1. a backbone with a bacterial selectable marker functional in both E. coli and A. tume- S 15 faciens spectinomycin resistance mediated by the aadA gene) and two origins of replication, functional in each of aforementioned bacterial hosts, plus the A. tumefaciens virG gene; 2. a plant-specific portion as described for biolistic vectors above, except in this instance this portion is flanked by A. tumefaciens right and left border sequences which mediate transfer of the DNA flanked by these two sequences to the plant.

5.2 Gene Silencing Vectors Vectors designed for reducing or abolishing expression of a single gene or of a family or related genes (gene silencing vectors) are also of two general types corresponding to the methodology used to downregulate gene expression: antisense or doublestranded RNA interference (dsRNAi).

Anti-sense For antisense vectors, a full-length or partial gene fragment (typically, a portion of the cDNA) can be used in the same vectors described for full-length expression, as part of the gene expression cassette. For antisense-mediated down-regulation of gene expression, the coding region of the gene or gene fragment will be in the opposite orientation relative to the promoter; thus, mRNA will be made from the non-coding (antisense) strand in planta.

dsRNAi For dsRNAi vectors, a partial gene fragment (typically, 300 to 500 basepairs long) is used in the gene expression cassette, and is expressed in both the sense and antisense orientations, separated by a spacer region (typically, a plant intron, eg. the OsSH1 intron 1, or a selectable marker, eg. conferring kanamycin resistance). Vectors of this type are designed to form a double-stranded mRNA stem, resulting from the basepairing of the two complementary gene fragments in planta.

Biolistic or binary vectors designed for overexpression or knockout can vary in a number of different ways, including eg. the selectable markers used in plant and bacteria, the transcriptional terminators used in the gene expression and plant selectable marker PF 56134 Scassettes, and the methodologies used for cloning in gene or gene fragments of inter- Sest (typically, conventional restriction enzyme-mediated or Gateway T recombinase- C1 based cloning).

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All publications, patents and patent applications are incorporated herein by reference.

While in the foregoing specification this invention has been described in relation to cer- PF 56134 I' tain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is suscepti- C(N ble to additional embodiments and that certain of the details described herein may be Svaried considerably without departing from the basic principles of the invention.

(U

Claims (8)

1. 2. The expression cassette of Claim 1, wherein the transcription regulating nucleotide sequence is selected from the group of sequences consisting of N i) the sequences described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 21, 24, 25, 28, 31, 32, 35, and 36, ii) a fragment of at least 50 consecutive bases of a sequence under i) which has substantially the same promoter activity as the corresponding transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, or 36; iii) a nucleotide sequence having substantial similarity with a sequence identity of at least 40% to a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, or 36; iv) a nucleotide sequence capable of hybridizing under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 2 X SSC, 0. 1% SDS at 50°C to a transcription regu- lating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, or 36, or the complement thereof; v) a nucleotide sequence capable of hybridizing under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 2 X SSC, 0. 1% SDS at 50"C to a nucleic acid com- prising 50 to 200 or more consecutive nucleotides of a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 15, 16, 17, 20, 21, 24, 25, 28, 31, 32, 35, or 36, or the complement thereof; vi) a nucleotide sequence which is the complement or reverse complement of any of the previously mentioned nucleotide sequences under i) to v).
2. 3. The expression cassette of Claim 1, wherein the functional equivalent of the tran- scription regulating nucleotide sequence is obtained or obtainable from plant ge- nomic DNA from a gene encoding a polypeptide which has at least 70% amino acid sequence identity to a polypeptide selected from the group described by SEQ ID NO: 5, 10, 14, 19, 23, 27, 30, and 34, respectively. PF 56134 87 S4. The expression cassette of any of Claim 1 to 3, wherein expression of the nucleic acid sequence results in expression of a protein, or expression of a antisense C RNA, sense or double-stranded RNA. S 5 5. The expression cassette of any of Claim 1 to 4, wherein expression of the nucleic acid sequence confers to the plant an agronomically valuable trait.
3. 6. A vector comprising an expression cassette of any of Claim 1 to
4. 7. A transgenic host cell or non-human organism comprising an expression cassette O 10 of any of Claim 1 to 5, or a vector of Claim 6. C 8. A transgenic plant comprising the expression cassette of any of Claim 1 to 5, a vector of Claim 6, or a cell of claim 7. S 15 9. A method for identifying and/or isolating a sequence with meristem-preferential or Smeristem-specific transcription regulating activity characterized that said identifica- tion and/or isolation utilizes a nucleic acid sequence encoding a amino acid se- quence as described by SEQ ID NO: 5, 10, 14, 19, 23, 27, 30, or 34 or a part of at least 15 bases thereof. The method of Claim 9, wherein the nucleic acid sequences is described by SEQ ID NO: 4, 9, 13, 18, 22, 26, 29, or 33, or a part of at least 15 bases thereof.
5. 11. The method of Claim 9 or 10, wherein said identification and/or isolation is realized by a method selected from polymerase chain reaction, hybridization, and database screening.
6. 12. A method for providing a transgenic expression cassette for meristem-preferential or meristem-specific expression comprising the steps of: I. isolating of a meristem-preferential or meristem-specific transcription regulating nucleotide sequence utilizing at least one nucleic acid sequence or a part thereof, wherein said sequence is encoding a polypeptide described by SEQ ID NO: 5, 10, 14, 19, 23, 27, 30, or 34, or a part of at least 15 bases thereof, and II. functionally linking said meristem-preferential or meristem-specific transcription regulating nucleotide sequence to another nucleotide sequence of interest, which is heterolog in relation to said meristem-preferential or meristem-specific transcription regulating nucleotide sequence. DATED this 9th day of December 2005. SunGene GmbH WATERMARK PATENT TRADEMARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VIC 3122 PF 56134 SEQUENCE LISTING <110> SunGene GmnbH <120> Expression cassettes for meristem-preferential expression in plants <130> AE20040926 PF 56134 AT <160> <170> Patentln version 3.3 <210> <211> <212> <213> <220> <221> <222> <223> 1630 DNA Arabidopsis thaliana promoter .(1630) transcription regulating sequence from Arabidopsis thaliana gene At 2gO 2180 <400> 1 tattgaaata ttgaacacat caggtttgaa aaacatatag c a aaat agt a tt t tggt tga atgaatagtc taaataaaat aataacaatt catttcgacc tttttaagaa ttgtataaga attagttgat agaccattaa atagtgtttt caatcacatt ccaattttaa a gaa tat ct t agttgaattt atatagtcac ctccaatggt tgacatatta taagcagttt cctaagatat aaatcagttg taaggctgca gtggatattt ttgaatgatt gatgaaagga atgagttgaa atttaacatt cgtaattcag attcattgtg atatcatttt tatgtattgc tatatacgta tcttgtaagc tgatgcatgg ttattgccac tacactaaat agattggaga gcctataaga atcatatttt acgtctttgg taagaactaa tagttttttt tgatttattt atcaatggaa atttcgttgg atgattgaca ttaaaaatgt gaaaatgttt ttgaatagtt tcaatttttc ttcaaccttt ctggaaattg tccaaattat gatcatacca tattaatcat aattcatatt tagattcttt cagagttaaa agttattaat aatccagatg tttcttccct aagtatatat gagtgcttca cacattgaca ttgattctta cttatgaaaa ttcagagttt gtaaaaaagg tcttttctag ttttagagca attatacgta gagctgaaca ttcctttaca aacatattca tattttaata ttcctccaca tcattttgat aatccacaaa agttgttttg gctatcaatc atgattgaaa caaaaaacaa gtagtggtat agattgtgaa catggaatga cacttacaaa accataattg ccaataacat actttgcatt catcttagct aattctaaaa taagtttgaa gaattcagga agatgaaaaa atgaatgtat ttattcaact gt tt gt tcat tttaaatggt caaccatttt aaataagaga ttactaatcc tcagctctat aaaataagat atagtttagt gccatatata taatttttct agtttagtat tttgtgatat ataagtccac caaaaaaatg cattttgatg cagggagaac taaattaaaa aagaaataaa tattagttta ccgagcaata tagatcactc gttgaaaata tttgtttcca cattcctctg tttttcatta aaaaagtgag cataataaat agaacgaatg attttgagaa ccattgctga ggatatcatt gtgtatattg aactcttaga taagaaaaac ggtggagatt aatcaatgat aagagacata aaaggaaaac atagatactg gttagaccat ttttaaaaca tcttattttc agatatacaa acttttctaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 PF 56134 ccgttgtgaa ttatttaatt agaaactccc cataattagc aaacggtaac ttttctaacc gttatgaatt attatttatt tcattcgaaa ctccccaaaa ttagcaaaca gtaacttttg taaaatataa tattttaccc gaacctgtaa cttattcgac cgttagaagt aatctctata tatacacctt 1500 1560 1620 1630 <210> 2 <211> 1631 <212> DNA <213> Arabidopsis thaliana <220> <221> <222> <223> promoter (1631) transcription regulating sequence from Arabidopsis thaliana gene At 2gO 2180 <400> 2 tattgaaata ttgaacacat caggtttgaa aaaacatata gcaaaatagt attttggttg gatgaatagt ttaaataaaa gaataaaaat acatttcgac atttttaaga tttgtataag gattagttga aagaccatta catagtgttt tcaatcacat tccaatttta aagaatatct cagttgaatt gatatagtca tctccaatgg atgacatatt ctaagcagtt acctaagata accgttgtga cgttatgaat gtaaaatata atatacacct aaatcagttg taaggctgca gtggatattt gttgaatgat agatgaaagg aatgagttga catttaacat tcgtaattca tattcattgt catatcattt atatgtattg atatatacgt ttcttgtaaa atgatgcatg tttattgcCa ttacactaaa aagattggag tgcctataag tatcatattt cacgtctttg ttaagaacta atagtttttt ttgatttatt tatcaatgga attatttaat tattatttat atattttacc t atttcgttgg atgattgaca ttaaaaaatg tgaaaatgtt attgaatagt atcaattttt tttcaacctt gctggaaatt gtccaaatta tgatcatac'c ctattaatca aaattcatat ctagattctt gcagagttaa cagttattaa taatccagat atttcttccc aaagtatata tgagtgcttc gcacattgac attgattctt tcttatgaaa tttcagagtt agtaaaaaag tagaaactcc ttcattcgaa cgaacctgta tcttttctag ttttagagca tattatacgt tgagctgaac tttcctttac caacatattc ttattttaat gttcctccac ttcattttga aaatccacaa tagttgtttt tgctatcaat tatgattgaa acaaaaaaca t gt agt ggt a gagattgtga tcatggaatg tcacttacaa aaccataatt accaataaca aactttgcat acatcttagc taattctaaa gtaagtttga ccataattag actccccaaa acttattcga gaattcagga agatgaaaaa aatgaatgta attattcaac agtttgttca atttaaatgg acaaccattt aaaataagag tttactaatc atcagctcta gaaaataaga catagtttag agccatatat ataatttttc tagtttagta atttgtgata aataagtcca acaaaaaaat gcattttgat tcagggagaa ttaaattaaa taagaaataa atattagttt accgagcaat caaacggtaa attagcaaac ccgttagaag tagatcactc gttgaaaata t ttt gttt cc tcattcctct ttttttcatt taaaaagtga tcataataaa aagaacgaat cattttgaga tccattgctg tggatatcat tgtgtatatt aaactcttag ttaagaaaaa tggtggagat taatcaatga caagagacat gaaaggaaaa gatagatact cgttagacca attttaaaac atcttatttt aagatataca aacttttcta cttttctaac agtaactttt taatctctat 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1631 PP 56134 <210> 3 <211> 1200 <212> DNA <213> Arabidopsis thaliana <220> <221> promoter <222> (1)..(1200) <223> transcription regulating sequence from Arabidopsis thaliana gene At 2gO 2180 <400> 3 gcctccatag cggtcaaaga ctattgtact aagattaatt agcctgcagc atgtttagaa caaaacccaa tttattattt cttccaaccg atggacattt atcgttggaa tatcgtcgaa ccaaaaaaat cctggccctg ctgtcattct t tct ct ttgt gcagcgtgtc ctccggttct atttttttat cagagagttc gatgctcatg caaaaatggt ccccaatcca cctacataat tccaaagtga tttttggaaa acctccaaaa attacaaatt ttattagatc tttcaacaat atctcttaca gctttttgta tttttatgca aagatatcta ctaatcataa gtggatcaag agtcaacttt cgtttccgac ttcgggaaaa tcattatgta ctgtctttga tccatttgac acacagcaac tagtattatc gaatcacgga atttctacgt attdttaatc atataaactg tacgacagcc tttttttgcg tacttacttc ggtatacgtt aaaacaaaat ttgtaagaaa tagcttactt ccagctttgg caacaactac gtcggccgtt taacaaaaaa tattcaaaat aggcccataa caagaagatg acacgaatgc ttctaacaat ttgtacttaa ttcgtagata tcgaattaat ttatatagtc gccaccacct tagaatccta cattaatctg tttttatttc gatctcatat gatcaattaa tgcttcactt tttgctcttt ttgactctcc tctttttcat agaaagaaat ctgaatcgag gtaaggccat atcatatagt ctagtcggtg tctcattttt atcgagtgcg taattttttg tatatttttt gattatacta tttctttttt acaagcagtt caaatcagct tctcacttta gaggactctt aaaagtaaaa gattaggcat atccacatgt cgacgataat ttttgtcgtt aaaaagaggt aaggaatctg gcctatttga attatgacag attcatcatg caaagattca aatatagctg ggattactag ttatcgtcga cgaacaattc catgcttttg ataattgtac aataaatttc aaaataaaaa gtatgttggg attaccataa aatatttgaa gccacgtgga taccggctat ttttttccaa gattcaaatc actcgtgtag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 <210> 4 <211> 1296 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (113)..(1024) <223> coding for tobamovirus multiplication protein 3 (TOM3) <400> 4 PF 56134 atttcgggaa aataacaaaa aaagaaagaa ataaaaagag tctcattatg tatattaaaa atctgaatag agaaggaata gtgattcaaa tccagagagt tgactagtgt ag atg aga Met Arg atc Ile ggc gga Gly Gly gta gag gtt aag aaa ttt gag tcg gag Val Glu Val Thr Lys Phe Ala Ser Glu tcg tct Ser Ser tcg tag gag gtg gag Ser Ser Ala Val Glu 10 atg Met ttg aat ctc Leu Asn Leu atg Met gaa Glu atg tag tcg Met Ser Ser gat tag aat Ala Ser Asn tgg Trp tgg taa gaa gta Trp Ser Asp Vai aaa Asn 40 gaa tat ccg att Glu Ser Pro Ile tgg Trp 45 gat agt ata tta Asp Arg Ile Phe aat gtt atc gat gtt atc taa gga ata His Val Leu Ala Vai Leu Tyr Gly Ile taa atc gtt gat Ser Leu Val Ala gtg att Val Ile aaa att gtg Gin Leu Val aaa aag gta Gin Lys Vai gtg gtg ttt Val Val Phe aga Arg ttt Phe ata aaa ttg aga Ile Gin Leu Arg gtt Vai .75 tta Phe gaa tat ggt Glu Tyr Gly cac ttt atc His Phe Leu aat Asn 90 aat Asn gtt gtt aat Val Val Asn gga Gly caa Gin tgg aag acg Trp Thr Thr gtt agt gat Val Arg Ala cca gag att Pro Giu Ile gta tta agg Val Phe Arg gtt aag ttt Val Gin Phe atg Leu 115 tat Tyr 100 caa Gin 166 214 262 310 358 406 454 502 550 598 646 694 742 790 aat ata ttg His Ile Leu att Leu 120 att Leu att cca agt Ile Pro Ser att Leu 125 att Ile tta tta aaa Phe Phe Thr gat att atg Ala Leu Leu tta tgg gat Phe Trp Ala gaa Glu 140 aga Ser tat tat aag gag agt Tyr Tyr Gin Ala Arg 145 gca gta tcg Ala Val Ser gtt gta tat Val Vai Tyr 165 act Thr 150 gga atc agg Gly Leu Arg tta tta aca Phe Phe Thr att aat gca Ile Asn Ala 160 gta gtt aag att Val Val Gin Ile gat Ala 170 cta Leu tgg ttg gtt Trp Leu Vai ttg tgg tgg aag Leu Trp Trp Lys 175 ttt gaa ggt gtt Phe Aia Gly Vai cct gtt Pro Vai 180 aga gtt atg gta Arg Vai Met Val ata Ile 185 tct aag atg Ser Lys Met tta Phe 190 ggt Gly tca Ser 195 ata Leu ttg tta gat gaa Leu Phe Aia Ala gga ttt tta att Gly Phe Leu Leu gga agg att Gly Arg Leu tta Phe 210 aag Lys atg ttg aaa agg ttt cca gta gaa tct Met Leu Gin Arg Phe Pro Vai Glu Ser ggg agg cga aaa Gly Arg Arg Lys PF 56134 ctg caa gag Leu Gin Giu gt t Val 230 at c Ile 215 ggt Gly tac gtg aca acc Tyr Val Thr Thr 235 220 ata tgc ttt acg Ile Cys Phe Thr tgt ttc ctc Cys Phe Leu 240 ggg gca aac Gly Ala Asn atc aga Ile Arg atg atg tgc Met Met Cys ttt Phe 250 gct gct ttc gat Ala Ala Phe Asp ctt gat Leu Asp 260 gtg tta gat cac Val Leu Asp His ccc Pro 265 atc ctt aac ttc Ile Leu Asn Phe gt a Val 275 cca Pro gag Giu ata tta ccc Ile Leu Pro tct ctg gtc ctc Ser Leu Val Leu ttc Phe 285 cag Gin ata tat tac ctg ttg Ile Tyr Tyr Leu Leu 270 atc ttg aga aag cta Ile Leu Arg Lys Leu 290 att cgc tga Ile Arg 838 886 934 982 1024 1084 1144 1204 1264 1296 cca aaa cga Pro Lys Arg ggc Giy 295 att aca caa tac Ile Thr Gin Tyr cat His 300 aatgtaaagg cacgacaact aatgatcaga tgaagaagac ttttctgtaa tcaatagaga gaaaagcata agatggaaat aaggcctttt ggttgatgat gggaatgqat tctacttgat gggtttgttc cctttatata aagqttatgt tgtaaaaaat gaattgtgta aatttatgtt gaatcatgtg gt aagactatgg gaaatagatc t t ttggt aaa ttattataaa ttgtttatgc taaagctgat cggtaaactg gtacatgtaa <210> <211> 303 <212> PRT <213> Arabidopsis thaiiana <400> Met Arg Ile Giy Giy Val Giu Vai Thr 1 5 Phe Ala Ser Giu Met Met Ser Ser Ser Ser Asn Trp Ser Ser Ser Ala Val Met Leu Asn Leu Lys Glu Ala Gin Asp Arg Trp Ser Asp Val As n Giu Ser Pro Ile Ile Phe His Val Leu Ala Leu Tyr Gly Ile Val Ser Leu Val Ala Ile Gin Leu Val Arg Ile Gin Leu Arg Pro Giu Tyr Gly Thr Thr Gin Lys Phe His Phe Leu Asn Phe Val Val Asn Gly Val PF 56134 Arg Ala Val Phe Val Phe Arg Arg 105 Asn Val Gin Phe Met Gin Pro 110 Ala Phe Phe Giu Ile Leu 115 Thr Thr Tyr 130 Gin His Ile Leu Leu 120 Asp Ile Pro Ser Leu 125 Ala Leu Leu Val1 135 Leu Phe Trp Ala Ile Tyr Tyr Gin Ala 145 Arg Ala Vai Ser Asp Giy Leu Arg Pro 155 Ser Phe Phe Thr Ile 160 Asn Ala Val Val Tyr 165 Val Val Gin Ile Leu Trp Leu Val Leu Trp 175 Trp Lys Pro Gly Val Ser 195 Arg Val Met Val Ile 185 Leu Ser Lys Met Phe Phe Ala 190 Gly Gly Arg Leu Phe Ala Ala Giy Phe Leu Leu Tyr 205 Leu Phe 210 Leu Met Leu Gin Arg 215 Phe Pro Val Giu Lys Giy Arg Arg Lys 225 Lys Leu Gin Giu Val 230 Giy Tyr Val Thr Thr 235 Ile Cys Phe Thr Cys 240 Phe Leu Ile Arg Ile Met Met Cys Phe 250 Ala Ala Phe Asp Glu Giy 255 Ala Asn Leu Leu Leu Val 275 Asp 260 Val Leu Asp His Pro 265 Ile Leu Asn Phe Ile Tyr Tyr 270 Ile Leu Arg Glu Ile Leu Pro Ser 280 Ser Leu Val Leu Lys Leu 290 Pro Pro Lys Arg Gly 295 Ile Thr GinTyr His 300 Gin Ile Arg <210> 6 <211> 510 <212> DNA <213> Arabidopsis thaliana PF 56134 <220> <221> pror <222> (1) <223> tral At5c 400> 6 atttctttaa caaatatatt tgtccttaaa acaaaattat tataaagtaa catactgtga ctatatatat cagatcaagc atttcgaatt roter .(510) iscription regulating sequence from Arabidopsis thaliana gene ;54 510 agagaataat ttattaacta ctattaaacc ataaatacca atcaaattaa aatgaccgtt gaactcttcc ttctttcacc ttaaacacaa ttaagttaaa aaataaagtg tttttattca ctttttgtaa acacgaatag ataccctttg tcttccattt attttcactc aacctaacgc tcagggggta ggagaattca agccagtacg tgaaacgaac taagccgttc atatcttctt ttcctcacac ttctttaagc gaaaaaatta agaatactat ggtattgacc taaaataaaa acttaaatcc caccctcttc ccttaaagct tttctttctt attcaaccat atagtttgaa aatatagaaa taaacgacat tggatttcaa ctctttcccc tcaacaaaac aatttctctc 120 180 240 300 360 420 480 510 <210> 7 <211> 511 <212> DNA <213> Arabidopsis thaliana <220> <221> promoter <222> (511) <223> transcription regulating sequence from Arabidopsis thaliana gene At 5g5 4510 <400> 7 atttccttta tcaaatatat atgtccttaa aacaaaatta ttataaagta acatactgtg tctatatata ccagatcaag catttcgaat aagagaataa tttattaact actattaaac tataaatacc aatcaaatta aaatgaccgt tgaactcttc cttctttcac tttaaacaca tttaagttaa aaaataaagt ctttttattc actttttgta aacacgaata tatacccttt ctcttccatt cattttcact aaacctaaac atcagggggt gggagaattc aagccagtac atgaaacgaa gtaagccgtt gatatcttct tttcctcaca cttctttaag agaaaaaatt aagaatacta gggtattgac ctaaaataaa cacttaaatc tcaccctctt cccttaaagc ctttctttct aattcaacca tatagtttga caatatagaa ataaacgaca ctggatttca cctctttccc ttcaacaaaa taatttctct <210> 8 <211> 396 <212> DNA <213> Arabidopsis thaliana <220> 1 PF 56134 <221> <222> <223> promoter transcription regulating sequence from Arabidopsis thaliana gene At g54510 <400> 8 atttctttaa caaatatatt tgtccttaaa acaaaattat tataaagtaa catactgtga ctatatatat agagaataat ttattaacta ctattaaacc ataaatacca atcaaattaa aatgaccgtt gaactcttcc ttaagttaaa aaataaagtg tttttattca ctttttgtaa acacgaatag ataccctttg tcttccattt tcagggggta ggagaattca agccagtacg tgaaacgaac taagccgttc atatcttctt ttcctc gaaaaaatta agaatactat ggtattgacc taaaataaaa acttaaatcc caccctcttc attcaaccat atagtttgaa aatatagaaa taaacgacat tggatttcaa ctctttcccc <210> 9 <211> 2181 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (115)..(1953) <223> coding for auxin-responsive GH3 protein <400> 9 acacccttaa agcttcaaca aaaccagatc aagcttcttt aagctttctt tcttaatttc tctcatttcg aattttaaac caccattttc actcttcttt acaaaaccta aacg atg Met cct gag gca Pro Glu Ala cca Pro aac Asn aag atc gca gct Lys Ile Ala Ala ttg Leu 10 caa Gln gag gtt tct gat Glu Val Ser Asp gct gag aag Ala Glu Lys aac gca gat Asn Ala Asp aag aac aaa Lys Asn Lys ctc Leu 25 cga Arg ttc atc gaa Phe Ile Glu gac Asp atc Ile gag agc ctc Glu Ser Leu gtg acc acg Val Thr Thr ctt tca cgt Leu Ser Arg gat gtt cag aga Asp Val Gln Arg gtt ctt gaa Val Leu Glu aat Asn gat Asp gct Ala cgt Arg gat gtg gag tat Asp Val Glu Tyr 55 gag act ttc aaa Glu Thr Phe Lys ctt Leu aaa cga cac Lys Arg His ggg Gly gtc Val gaa gga cga Glu Gly Arg cat atc atg His Ile Met gta act tac Val Thr Tyr gaa gat Glu Asp caa gtc att caa cct gag atc aac aga atc gcc ggt gat aag tct PF 56134 Ile Gin Pro Giu Ile Asn Arg Ile otc tgt tct Leu Cys Ser 100 aac Asn Ala 90 tto Phe 000 ato tct Pro Ile Ser otc aoa agt Leu Thr Ser tot Ser 110 ggg act tot Gly Thr Ser ggt gga Gly Gly 115 Asn Gly Asp Lys Ser Gin Val gag agg aaa ctg Glu Arg Lys Leu atg Met 120 otc Leu aca ato gaa Thr Ile Glu gaa ota gao aga Glu Leu Asp Arg aga Arg 130 coct Pro tca Ser ott otc tao Leu Leu Tyr agt Ser 135 ggo Gly ttg atg coct Leu Met Pro gtg Val 140 atg gac cag ttt Met Asp Gin Phe gtt Vai 145 ggt ott gao Gly Leu Asp gaa too aag Glu Ser Lys aaa Lys 150 oca Pro tot Ser aaa ggg atg Lys Giy Met ttt otg ttt ato Phe Leu Phe Ile aaa toa Lys Ser 160 ggt ggt tc coct Gly Gly Leu Pro 170 cac tto aaa aao His Phe Lys Asn ogt cot gtt tta aco agt Arg Pro Vai Leu Thr Ser 175 cct tat gat cot tac aco Pro Tyr Asp Pro Tyr Thr tac tao aaa Tyr Tyr Lys 180 aac tao aca Asn Tvr Thr aga Arg agt cc aao Ser Pro Asn 195 oaa Gin 200 ott Leu ato ctt tgt Ile Leu Cys tot Ser 205 oaa Gin 190 gao Asp tot tao oag Ser Tyr Gin ago Ser 210 ott Leu atg Met ogt Arg tao tot caa Tyr Ser Gin gtt ggt got Vai Gly Ala atg Met 215 tgt ggt tta Cys Gly Leu tgc Cys 220 ttc Phe cac aaa gag His Lys Glu gtt Vai 225 453 501 549 597 645 693 741 789 837 885 933 981 1029 1077 1125 ttt ott gag Phe Leu Glu aaa Lys 245 too Ser 230 cat His gag Glu gtt ttt gc tct Val Phe Ala Ser tgg oct gag cta Trp Pro Giu Leu 250 ata aoo gat tct Ile Thr Asp Ser cgt gao att Arg Asp Ile aga Arg 255 gcg Ala att aga gc Ile Arg Ala ato aag Ile Lys 240 aco ggt Thr Gly gtc ggg Val Gly act otc agt Thr Leu Ser 260 tcg gtt ogt Ser Val Arg gag Glu 270 gto Val gag att Glu Ile 275 tgc agg Cys Arg 290 ott aaa cog gat Leu Lys Pro Asp aag act tot tgg Lys Thr Ser Trp 295 tat gtg gat gtg Tvr Val AsD Val cct Pro 280 oaa Gin ott got gat Leu Ala Asp ggg ato ato Gly Ile Ile act Thr 300 aca Thr agg ott tgg oca Arg Leu Trp Pro gaa tot gaa Glu Ser Glu act aag Thr Lys cca act otg gat tao att gtg act Ile Vai Thr ago aat ggt gga Gly 315 ttg atg toa cag Met Ser Gin tat att Tyr Ile 320 aca atg cct ott gtc tgo PF 56134 Pro Thr Leu tat gct tct Tyr Ala Ser 340 aaa cca agt Lys Pro Ser 355 Tyr Tyr Ser Asn Gly Leu 330 ggt gtg Gly Val Pro Leu Val gag tgt tac Glu Cys Tyr ttc Phe 345 aat ctc agg Asn Leu Arg 350 ccg aac atg Pro Asn Met Cys Thr Met 335 cca ctc tgc Pro Leu Cys gcg tat ttc Ala Tyr Phe gaa gtc tct Glu Val Ser act ctc ata Thr Leu Ile 365 act Thr gag Glu 370 ctt Leu ttc ttg cct gtt Phe Leu Pro Val agg aac agt gga Arg Asn Ser Gly agc tct atc Ser Ser Ile agt Ser 385 cca aaa gca Pro Lys Ala ctc Leu 390 act gag aaa gaa Thr Glu Lys Glu caa Gin 395 gag ctt gtt Glu Leu Val gat ctc Asp Leu 400 gtc gat gtc Val Asp Val gct ggg ctt Ala Gly Leu 420 ttc aag aac Phe Lys Asn ctt ggt cag gag Leu Gly Gin Glu agg tac aga Arg Tyr Arg gtg Val 425 ttc Phe tac gag Tyr Glu 410 ggt gat Gly Asp agc ttc Ser Phe ctt gtt gtc Leu Val Val gtc cta agc Val Leu Ser 430 ata tgc cgc Ile Cys Arg 445 acc acc tat Thr Thr Tyr 415 gtg get ggt Val Ala Gly aag aac gtg Lys Asn Val aat gcg cct Asn Ala Pro cag Gin 440 435 tta Leu gtc Val 450 gca Ala agc att gac Ser Ile Asp gac aaa acc gat Asp Lys Thr Asp gtt gag ctt caa Val Glu Leu Gin aac Asn 465 1173 1221 1269 1317 1365 1413 1461 1509 1557 1605 1653 1701 1749 1797 1845 gtt aaa aac Val Lys Asn gcg Ala 470 gta aca cac ctt Val Thr His Leu ttt gat gct Phe Asp Ala tca ctc Ser Leu 480 cac tat His Tyr tec gag tac Ser Glu Tyr ago tat gcg gac Ser Tyr Ala Asp aca Thr 490 aac Asn tot atc cog Ser Ile Pro gtc tta ttc Val Leu Phe 500 tcg gtc ttc Ser Val Phe gag ctc tgc Glu Leu Cys ggt aac acg Gly Asn Thr cca att cct ccc Pro Ile Pro Pro 510 tca ctt aac agt Ser Leu Asn Ser gag gat tgc Glu Asp Cys tgt Cys 520 gtc Val tta acc ata gag Leu Thr Ile Glu gtg Val 530 atc Ile aga caa gga Arg Gin Gly agt gat aag Ser Asp Lys tcc Ser 540 aag Lys gga cca ttg Gly Pro Leu aag atg gtc Lys Met Val gag Glu 550 ggg act ttc Gly Thr Phe ctc atg gat Leu Met Asp tat gcg Tyr Ala 560 tgt gtg ata ago ttg ggt gca tcg ate aat cag aag aca cca agg PF 56134 Ile Ser Leu Gly Ala Ser Ile Asn Gin Tyr Lys Thr Pro Arg Cys Val 565 570 575 aag ttt got cog atc att gag ctt tta aac tct agg gtt gtt gat agt Lys Phe Ala Pro Ile Ile Giu Leu Leu Asn Ser Arg Val Val Asp Ser tac ttc Tyr Phe 595 580 ago Ser ccc aag tgt Pro Lys Cys cct Pro 600 aaa tgg too cct ggt cac aag caa tgg Lys Trp Ser Pro Gly His Lys Gin Trp 585 1893 1941 1993 ggg agt aac taa agaggaaact ttggggaaga agaaagactO tctatgaagt Gly Ser Asn 610 agaaggttot gttttgtaat caaatgaata togagaaaag ttgttotaat ttaaatotta atttaatttt gctttactgt agattotagt catatgtaca tagcoggttt atgtttotct tocagcct tgataaatta ttatgtotgt ttttgttttg aaatatgttt aagcgactct tttaagttto 2053 2113 2173 2181 <210> <211> 612 <212> PRT <213> Arabidopsis thaliana <400> Met Pro Glu Ala Pro 1 Lys Ile Ala Ala Leu Glu Val Ser Asp Glu Ser Leu Ala Glu Thr Asn Ala Lys Asn Lys Asn Lys Leu 25 Gin Phe Ile Glu Asp Val Thr Ile Leu Ser Asp Asp Val Gin Arq Arg Val Leu Glu Arg Asn Ala Asp Val Glu Leu Lys Arq His Gly Leu Giu Giy Arg Thr Asp Arg Giu Thr Phe Lys His Ile Met Val Val Thr Tyr Asp Ile Gin Pro Ile Asn Arg Ile Ala Asn Gly Asp Lys Ser Gin Val Leu Cys Ser Gly Gly 115 Ser 100 Asn Pro Ile Ser Phe Leu Thr Ser Ser Gly Thr 110 Glu Leu Asp Glu Arg Lys Leu Met 120 Pro Thr Ile Glu PF 56134 Arg Arg 130 Ser Leu Leu Tyr Ser 1.35 Leu Leu Met Pro Met Asp Gin Phe Vai 145 Pro Gly Leu Asp Lys 150 Giy Lys Giy Met Tyr 155 Phe Leu Phe Ile Ser Giu Ser Lys Thr 165 Pro Gly Gly Leu Pro 170 Aia Arg Pro Vai Leu Thr 175 Ser Tyr Tyr Thr Asn Tyr 195 Ser Ser His Phe Asn Arg Pro Tyr Asp Pro Tyr 190 Asp Ser Tyr Thr Ser Pro Asn Gin 200 Thr Ile Leu Cys Ser 205 Gin Ser 210 Met Tyr Ser Gin Leu Cys Giy Leu Gin His Lys Glu Val 225 Leu Arg Vai Giy Aila 230 Val Phe Aia Ser Phe Ile Arg Aia Lys Phe Leu Giu His Trp Pro Giu Leu 250 Ala Arg Asp Ile Arg Thr 255 Giy Thr Leu Giy Giu Ile 275 Ser Giu Ile Thr Asp 265 Ser Ser Vai Arg Giu Aia Val 270 Val Glu Ser Leu Lys Pro Asp Lys Leu Aia Asp Giu Cys 290 Arg Lys.Thr Ser Trp 295 Gin Giy Ile Ile Thr 300 Arg Leu Trp Pro Asn 305 Thr Lys Tyr Vai Asp 310 Val Ile Vai Thr Giy 315 Thr Met Ser Gin Ile Pro Thr Met Tyr Ala Cys Lys Pro 355 Leu Asp 325 Ser Ser 340 Tyr Tyr Ser Asn Giy 330 Leu Pro Leu Vai Cys Thr 335 Pro Leu Giu Cys Tyr Giy Val Asn Leu Ser Giu Val Ser T yr 360 Thr Leu Ile Pro Met Ala Tyr PF 56134 Phe Giu Phe Leu Pro Val His Arg Asn Ser Giy 370 375 Val 380 Thr Ser Ser Ile Ser 385 Leu Pro Lys Ala Leu 390 Thr Giu Lys Giu Gin Gin Giu Leu 395 Giu Leu Val. Val Val Leu Val Asp Val Lys 405 Leu Gly Gin Giu Tyr 410 Thr Thr 415 Tyr Ala Gly Giy Phe Lys 435 Tyr Arg Tyr Arg Gly Asp Val Leu Ser Val Ala 430 Arg Lys Asn Asn Asn Aia Pro Phe Ser Phe Ile Val Val 450 Leu Ser Ile Asp Ser 455 Asp Lys Thr Asp Giu 460 Val Giu Leu Gin Asn 465 Ala Vai Lys Asn Val Thr His Leu Val 475 Pro Phe Asp Ala Leu Ser Giu Tyr Thr 485 Ser Tyr Ala Asp Ser Ser Ile Pro Gly His 495 Tyr Val Leu Pro Ser Vai 515 Phe 500 Trp Giu Leu Cys Leu 505 Asn Gly Asn Thr Pro Ile Pro 510 Ser Leu Asn Phe Giu Asp Cys Cys 520 Leu Thr Ile Giu Ser Vai 530 Tyr Arg Gin Giy Arg 535 Vai Ser Asp Lys Ser 540 Ile Gly Pro Leu Giu 545 Ile Lys Met Vai Ser Gly Thr Phe Asp 555 Lys Leu Met Asp Tyr 560 Ala Ile Ser Leu Giy 565 Ala Ser Ile Asn Tyr Lys Thr Pro Arg Cys 575 Vai Lys Phe Ser Tyr Phe 595 Ala 580 Pro Ile Ile Giu Leu 585 Leu Asn Ser Arg Val Val Asp 590 His Lys Gin Ser Pro Lys Cys Pro 600 Lys Trp Ser Pro Gly 605 PF 56134 Trp Gly Ser Asn 610 <210> 11 <211> 2552 <212> DNA <213> Arabidopsis thaliana <220> <221> <222> <2 23> promoter (2552) transcription regulating sequence from Arabidopsis thaliana gene At 2g2 6970 <400> 11 tgttttggga ttgatgttag ttttacgatg aatgacatgg ctaatctttt ataaacaaaa accaaaaggg ttaatatatt tttgggtaca tagagaacta cctacatgac caaattctag aatgttcttt agaggggttz agcgacaatg gcctgtcttg acgtccacga gacgatgaga ctccgtgttc atttaccatt ttctgaaatt ttcttgttat aaactttgtg ttcagttatt tggaagaaaa cgtagtggaa aacattgtgt caattcatca cgaagactta gatgcattca gctttaaacg aagatccttt atagtcatga aagtcttgat tgttgactgt tgaatatcac ttagctcttc tacaacgata atcgagtgat tacatcatct tcacaaaata ccaagtaaaa ttgaaagcag aaaactccac agtgtgtaga aggatcgaag catagaacgt tttccccaga gatgatggtg ttttgtttgg catgtggtct tagtacaagt caacttattt acgatttaca tttaacaagt taagcttatg cttgcaaaac tcaaaatata ataaagaagc ttcaacatca tgacacacaa aattaatagt gaagaaacag cacacatgaa atatggatta t tgt ct tcgt tatgtgtttt ggacggcgtt acaataaaat tcaaaattca taaagagtga tgcttccgaa cgttaaagga ctgcggtctg aatgggcaag acatgccgag agaaagtcct atgatggtgg gacaaaactt cttaaatgtg agaagtcaat tcatgatata tcatttgaac tattcttcta agtttcaagt tctaaacata aaaatgtaaa cttcttgata ttacagcacc aataaataaa cacaaatggt atgatggtat tttaaattgt agtttgtctt gtgtttttct gttgatcaat tgttatttaa gcaaaaatgg aagaaacgca tcatagttgt gagcatgaaa cataacggcg aacgagCCCC ccactcgacg acttgtcccg atgcatcatg catgttacca agagaaaatg tttgtggttg actaatttta aatggtaaca ataatttgag aacgatattt tcagttcaat gaccaaagaa tttggctcaa aacagagatc cgttttgttt atattgaagg ttcttcttct attgtgaact tatggattta agatatctat tttactttct aaaaagttca t t tgt ggtt g ctatattctt aattaaatac tcgaagggtt cccacggcga. agcatcacga ttggcgcggC ataacagcaa ggccagccgg atcatgttgg tgatccatta gtttcgtatt tactcacgtt gaaagccttt caccacttgt aaagtgatga acttataaat caacaaaatg attaggcaaa aaccttggac ct tgt gagt t tgtcttttct aagttccaaa tattcttcgt aatatatgga tatctacgaa acaacgaagt tataaatttt ccttatctcg aaatttatca cataaaattC acacaaacga cctcgtcatc atccggcgat gataagcaag tagtggagcc ggaggaagac agaagagtat aattggtatg tatgataagt tattgtgatg ttgacttgtc taaataccta agaattgtgg aatttatatt caatgacaac caactctccc cccaaaccta aagaaacata 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 PF 56134 tcaatctagg ggacaaaacg aaacaaatat tctcaagtgt gtaatctcaa acattagtca tatgttct tt t aagt ttat t cgattttttc caaactctcc ttcttcttcc gtttagattt gtcaagagtc tcttgtggct tctaaaattc aaccaataca acaagatata tagtcgcaaa gtgttagtcg atttctatac ttactgacta tcgccgtttt tcttttaggt aacgctttct actggtatcc ttgagactga aattaaagta acaagatttg actggttcgt gacatatagt taaaaaagtt tgtggtcaca gaaatgtggt aaaattaaat aaatagttat tttttttatt taccttcctc ctgtcctggc cctgtttccC atttgataat ttgaccttta ttggtttgag cacaacccat ggatacaacc ttctaatata gttatgggat cacaattatg ctatctaatt aattatattt tgtttggctt cgattagtct tttcgccgat tcttccgatt atattgacta aactttagtt ac tctagaaaat atagaaatgt cgatatcaaa tgtaaggtag agattacttt attttttgac t t taagt aaa tgtttttggg tactgaaaga gaagatgctc tgattacaac aattaatcag tttttactca tcgaaaacaa tatcaagatc tcagttgtaa caaatcagtt gcgaattgcg caatatggta acatgttaat tcaaagagac aaaaaatgaa caatggcttC acaaaaccat tgatttagag tggcactgtc 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2552 <210> <211> <212> <213> <220> <221> <222> <223> 12 2552 DNA Arabidopsis thaliana promoter (1)..(2552) transcription regulating sequence from Arabidopsis thaliana gene At 2g2 6970 <400> 12 tgttttggga ttgatgttag ttttacgatg aatgacatgg ctaatctttt ataaacaaaa accaaaaggg ttaatatatt tttgggtaca tagagaacta tctacatgac caaattctag aatgttcttt agaggggttc agcgacaatg gcctgtcttg acgtccacga gacgatgaga gatgcattca gctttaaacg aagatccttt atagtcatga aagtcttgat tgttgactgt tgaatatcac ttagctcttc tacaacgata atcgagtgat tacatcatct tcacaaaata ccaagtaaaa ttgaaagcag aaaactccac agtgtgtaga aggatcgaag catagaacgt ataaagaagc ttcaacatca tgacacacaa aattaatagt gaagaaacag cacacatgaa atatggatta ttgtcttcgt tatgtgtttt ggacggcgtt acaataaaat tcaaaattca taaagagtga tgcttccgaa cgttaaagga ctgcggtctg aatgggcaag acatgccgag cttcttgata ttacagcacc aataaataaa cacaaatggt atgatggtat tttaaattgt agtttgtctt gtgtttttct gttgatcaat tgttatttaa gcaaaaatgg aagaaacgca tcatagttgt gagcatgaaa cataacggcg aacgagcccc ccactcgacg acttgtcccg aacagagatc cgttttgttt atattgaagg ttcttcttct attgtgaact tatggattta agatatctat tttactttct aaaaagttca tttgtggttg ctatattctt aattaaatac tcgaagggtt cccacggcga agcatcacga ttggcgcggc ataacagcaa ggccagccgg cttgtgagtt tgtcttttct aagttccaaa tattcttcgt aatatatgga tat ct acgaa acaatgaagt tataaatttt ccttatctcg aaatttatca cataaaattc acacaaacga tctcgtcatc at ccggcgat gataagcaag tagtggagcc ggaggaagac agaagagtat 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 PF 56134 ctccgtgttc atttaccatt ttctgaaatt ttcttgttat aaactttgtg ttcagttatt tggaagaaaa cgtagtggaa aacattgtgt caattcatca cgaagactta tcaatctagg ggacaaaacg aaacaaatat tctcaagtgt gtaatctcaa acattagtca tatgttcttt taagtttatt ccgatttttt acaaactctc cttcttcttc tgtttagatt ggt caagagt ctcttgtggc tttccccaga gatgatggtg ttttgtttgg catgtggtct tagtacaagt caacttattt acgatttaca tttaacaagt taagcttatg cttgcaaaac tcaaaatata tctaaaattc aaccaataca acaagatata tagtcgcaaa gtgttagtcg atttctatac ttactgacta tcgccgtttt ctcttttagg caacgctttc cactggtatc tttgagactg caattaaagt tacaagattt agaaagtcat atgatggtgg gacaaaactt cttaaatgtg agaagtcaat tcatgatata tcatttgaac tattcttcta agtttcaagt tctaaacata aaaatgtaaa *actggttcgt gacatatagt taaaaaagtt tgtggtcaca gaaatgtggt aaaattaaat aaatagttat ttttttttat ttaccttcct tctgtcctgg ccctgtttcc aatttgataa attgaccttt gttggtttga atgcatcatg catgttacca agagaaaatg t t tgt ggtt g actaatttta aatggtaaca ataatttgag aacgatattt tcagttcaat gaccaaagaa tttggctcaa cacaacccat ggatacaacc ttctaatata gttatgggat cacaattatg ctatctaatt aattatattt ttgtttggct ccgattagtc ctttcgccga ctcttccgat tatattgact aaactttagt ga atcatgttgg tgatccatta gtttcgtatt tactcacgtt gaaagccttt caccatttgt aaagtgatga acttataaat caacaaaatg attaggcaaa aaccttggac tctagaaaat atagaaatgt cgatatcaaa tgtaaggtag agattacttt attttttgac tttaagtaaa ttgtttttgg ttactgaaag tgaagatgct ttgattacaa aaattaatca aattggtatg tatgataagt tattgtgatg ttgacttgtc taaataccta agaattgtgg aatttatatt caatgacaac caactctccc cccaaaccta aagaaacata tcgaaaacaa tatcaagatc tcagttgtaa caaatcagtt gcgaattgcg caatatggta acatgttaat gtcaaagaga aaaaaaatga ccaatggctt cacaaaacca gtgatttaga 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2552 ttttttactc atggcactgt <210> 13 <211> 828 <212> DNA <213> Arabidopsis thaliana <220> <221> <222> <223> CDS (680) coding for exonuclease family protein <400> 13 gaaagaaaaa a atg aac aaa ctc tcc aac gct ttc tct gtc ctg gct ttc Met Asn Lys Leu Ser Asn Ala Phe Ser Val Leu Ala Phe 1 5 gcc gat gaa gat gct cca atg gct tct tct tct tcc act ggg aaa caa Ala Asp Glu Asp Ala Pro Met Ala Ser Ser Ser Ser Thr Gly Lys Gln 20 gaa gaa agt gta aat ggg tca ctt gag gat gga gat tac aag caa cca Glu Glu Ser Val Asn Gly Ser Leu Glu Asp Gly Asp Tyr Lys Gln Pro 98 146 PF 56134 ctt gtt tgg att Leu Val Trp Ile gac Asp ttg gaa atg act Leu Glu Met Thr tta Leu aat gtt gaa Asn Val Giu gtt gac Val Asp aca caa Thr Gin agg ata ttg Arg Ie Leu tca gtg gag Ser Val Glu gag Giu ggt Gly att gca tgt ata Ile Aia Cys Ie att Ile 70 aat gga gat Asn Giy Asp tta Leu cca gat tta Pro Asp Leu gta cgt caa acg Vai Arg Gin Thr aaa Lys gac tgt ttg Asp Cys Leu gat aaa Asp Lys atg gat gac tgg Met Asp Asp Trp tgt Cys 100 agt Ser caa act cat cat Gin Thr His His gctagt ggg ttg Aia Ser Gly Leu acg Thr 110 aag Lys aag Lys aaa gtg ctc Lys Vai Leu gcg ata act Ala Ile Thr gaa Giu 120 ggt Gly gaa gct gag Giu Ala Glu caa Gin 125 194 242 290 338 386 434 482 530 578 626 gtc atc gaa Val Ile Giu aag aag cat Lys Lys His tcc gga aat Ser Gly Asn cca ctg Pro Leu 140 tta gct gga Leu Ala Gly atg cca gaa Met Pro Giu 160 agc gtc aag Ser Val Lys aac As n 145 tta Leu gtc tat gtc Val Tyr Val gat Asp 150 cct Pro ctt ttc tta Leu Phe Leu gct gcc ctt Ala Ala Leu ttc Phe 165 cga Arg cat ata ctc His Ile Leu gt c Val 170 gag Giu Lys Lys Tyr 155 gat gtc agt Asp Val Ser aga agg aaa Arg Arg Lys gct tta tgc Ala Leu Cys tgg ttc ccc Trp Phe Pro gct Ala 190 agt Ser aga Arg 175 cct Pro gcc aag aaa Ala Lys Lys aac As n 195 aag Lys cac aga gcc His Arg Ala atg Met 200 aca Thr gat ata aga gaa Asp Ile Arg Giu 205 ttc aaa gct agg Phe Lys Ala Arg ata aag gag Ile Lys Giu tac tac aag Tyr Tyr Lys aaa Lys 215 at a Ile 220 tga gtggagttgg gtactgcaat atgcttactt actagttaga aggatccgtt ttagttttac gtattggtct gaattcggca ttgttattct cttgacgatt gtatcctcaa aacctaaatt cttgataaag aaaaaccttt gaagatgg <210> <211> <212> <213> 14 222 PRT Arabidopsis thaliana PF 56134 <400> 14 Met Asn Lys Leu Ser 1 5 Asn Ala Phe Ser Val 10 Leu Ala Phe Ala Asp Giu Asp Ala Pro Val Asn Gly Met Ala Ser Ser Ser Ser 25 Thr Gly Lys Gin Glu Glu Ser Ser Leu Glu Asp Gly 40 Asp Tyr Lys Gin Pro Leu Val Trp Ile Asp Leu Glu Met Thr Leu Asn Val Giu Val Asp Arg Ile Leu Glu 65 Ile Ala Cys Ile Ile 70 Thr Asn Gly Asp Leu Thr Gin Ser Vai Gly Pro Asp Leu Vai Val Arg Gin Thr Lys 90 Asp Cys Leu Asp Lys Met Asp Asp Trp, Val Leu Leu 115 Cys 100 Gin Thr His His Ala Ser Gly Leu Thr Lys Lys 110 Lys Val Ile Ser Ala Ile Thr Giu 120 Arg Giu Ala Giu Giu Phe 130 Val Lys Lys His Gly Ser Gly Asn Pro 140 Leu Leu Ala Gly Asn 145 Ser Val Tyr Val Asp 150 Phe Leu Phe Leu Lys Tyr Met Pro Leu Ala Ala Leu Pro His Ile Leu Val 170 Asp Val Ser Ser Val Lys 175 Ala Leu Gys Lys Lys Asn 195 Al a 180 Arg Trp Phe Pro Giu Arg Arg Lys Ala Pro Ala 190 Ser Ile Lys Asn His Arg Ala Met 200 Asp Asp Ile Arg Giu Leu 210 Lys Tyr Tyr Lys Lys 215 Thr Ile Phe Lys Ala Arg Arg 220 <210> <211> 2193 PF 56134 <212> DNA <213> Arabidopsis thaliana <220> <221> <222> 223> promoter (1)..(2193) transcription regulating sequence from Arabidopsis thaliana gene At2g01180.. <400> ataatcaacc gaaacacgat aagttataag agctgttgga aaagaccatt taattttgag agagagacgt gaaatgcaaa ataaaatatt tttggtaact ttgacattct taaaactata tgtgcaagtt tataaaaaac tttgtgattg caaaaccaaa ttttgcccta cacacatcat tgtttccaat aattccattt atatctggat tgagaaaagg gtcaatagaa aaatttcgtt agcaagttgc gacttttcga tagtttccac atttgttgta ctggatatgt aaccccctat taaggcctaa accttcctaa gcgataactg ct ctct gtt t gcaagtttaa ttctcgtttg taaaaggaga aagaggtgac aagacaggcg ctacagaatt tttgtatatc aatgatgatg agtgaaacaa taaatcattt ttttttggag ctctttctct tatttatatc ttttagctat tgttaaaaaa gtaaatgtca accttaaaaa tcaaattaaa taacaaaaaa ttttaagtcc ccacccatac tccagctttt tctaattgcg gcaagataat ttacaataaa catgcactct cctcattatg tttcttcctc ttctaaaaag gtgtaatgga gtagtttgaa taagtaaggt aaagtgggac aaaagtcaaa tcttcttatt caacgcgtct atcgtgattt tttcaagaaa ttgactacat taaagtttgt gacgtgttaa ttctctcttt tttgtgcatt ggaaacctaa taaaatttaa accgtgatgt ctattgtcct cataaaatat taattttatc agctgagaat aactatttat ttaaagccgt attggtgtgt aaaaaatcac accaccaaac acaattatag aagtaggatt tgctccataa ttgaacgtcc caaagaaagg ccaaaataaa tacttactta aataagttcc ttatatattc tacatattag aactgaatca aagcccacag ctacgcggct agtcaaaatt cttctatcaa tctacctcct ggtttatgtt agcccttata tttatggttg gtaatgtata tgtgtatatc ttcttacgat accgagtgag aagagtaagc ttaatattaa cgtcaaaaca tctttttttt ataaaagaaa ggaatagtgc aaaatttttt actgttttaa tatgctattt tgatgagtgc acataaaagt tggacccact tagtccacct at tat.t ggt t acccttattc aagtcatcat ttccgagtcc ataaaaaaca cttcatcttc atttttggat tatttgctgg taaatagtta tctgacaaaa agaaatatac gagaagaaag caagaggaag accgtcaagt cctcatcgga tgtcgatctc ataagaactc ggaatgaaat aacagaatct tggaattagg caatatgcaa ataactaaat aaacaaacaa ttcgttttta ttcaagatgt cttcttttac tcttattagt aaaactgtta ataacacttt agtgttaagt ttttaataaa aaaagcgtag gtaattgtga aacccctact tttttctttt ggtaaaagat tagttcaacc catctttttt tcaagcatct gacaaagact tatttggcac tctttcctat gctttagata agtccaaaaa cgagcaggga ttacgggctt acgaaggcca aggagtttct tgggaaacaa ctatttatct atattataca tatgaaacct tcgaagatct cttttgtacg tattaaacat attagcaaat gatgtttcaa caaatcagca ctttaaggtt atattgatta t t t ttaact t aactaaaagt gaaaaaactg tggtaacaat ggttaccatt gaagacatga gcaaataaat ggaaaaaaga cagggtatac ctttctacac gcaaagaata ataattgtca tttctttctg ctcacgtcgg ttagacttgt ccacaaacag ttttacacaa tgggctggct agaaattata aggcccataa ggatttccga tatcagaccg aatcatgtct aacaaaactc atagtcttcg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 PF 56134 ttagatcaat tcctctgttt tactcctaag tttcgagatc cacatttctc tttaacctca 2160 tctcatctct tagtcgagat cttcactttc tgc 2193 <210> <211> <212> 213> <220> <221> <222> <223> 16 2192 DNA Arabidopsis thaliana promoter (1)..(2192) transcription regulating sequence from Arabidopsis thaliana gene At 2 <400> 16 ataatcaacc ttctcgtttg atcgtgattt ggtttatgtt gaaacacgat aagttataag agctgttgga aaagaccatt taattttgag agagagacgt gaaatgcaaa ataaaatatt tttggtaact ttgacattct taaaactata tgtgcaagtt tataaaaaac tttgtgattg caaaaccaaa ttttgcccta cacacatcat tgtttccaat aattccattt atatctggat tgagaaaagg gtcaatagaa aaatttcgtt agcaagttgc gacttttcga tagtttccac atttgttgta ctggatatgt aaccccctat taaaaggaga aagaggtgac aagacaggcg ctacagaatt tttgtatatc aatgatgatg agtgaaacaa taaatcattt ttttttggag ct ctt tct ct tatttatatc ttttagctat tgttaaaaaa gtaaatgtca accttaaaaa tcaaattaaa taacaaaaaa ttttaagtcc ccacccatac tccagttttt tctaattgcg gcaagataat ttacaataaa catgcactct cctcattatg tttcttcctc ttctaaaaag gtgtaatgga gtagtttgaa tttcaagaaa ttgactacat taaagtttgt gacgtgttaa ttctctcttt tttgtgcatt ggaaacctaa taaaatttaa accgtgatgt ctattgtcct cataaaatat taattttatc agctgagaat aactatttat ttaaagccgt attggtgtgt aaaaaatcac accaccaaac acaattatag aagtaggatt tgctccataa ttgaacgtcc caaagaaagg ccaaaataaa tacttactta aataagttcc ttatatattc tacatattag aactggatca agcccttata t ttat ggt tg gtaatgtata tgtgtatatc ttcttacgat accgagtgag aagagtaagc ttaatattaa cgtcaaaaca tctttttttt ataaaagaaa ggaatagtgc aaaatttttt actgttttaa tatgctattt tgatgagtgc acataaaagt tggacccact tagtccacct attattggtt acccttattc aagtcatcat ttccgagtcc ataaaaaaca cttcatcttc atttttggat tatttgctgg taaatagtta tctgacaaaa tgtcgatctc ataagaactc ggaatqaaat aacagaatct tggaattagg caatatgcaa ataactaaat aaacaaacaa ttcgttttta ttcaagatgt cttcttttac tcttattagt aaaactgtta ataacacttt agtgttaagt ttttaataaa aaaagcgtag gtaattgtga aacccctact t tt t tct tt t ggtaaaagat tagttcaacc catctttttt tcaagcatct gacaaagact tatttggcac tctttcctat gctttagata agtccaaaaa cgagcaggga atattataca tatgaaacct tcgaagatct cttttgtacg tattaaacat attagcaaat gatgtttcaa caaatcagca ctttaaggtt atattgatta tttttaactt aactaaaagt gaaaaaactg tggtaacaat ggttaccatt gaagacat ga gcaaataaat ggaaaaaaga cagggtatac ctttctacac gcaaagaata ataattgtca tttctttctg ctcacgtcgg ttagacttgt ccacaaacag ttttacacaa tgggctggct agaaattata aggcccataa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 -I~ PF 56134 taaggcctaa accttcctaa gcgataactg ctctctgttt gcaagtttaa ttagatcaat tctcatctct. taagtaaggt aaagtgggac aaaagtcaaa tcttcttatt caacgcgtct tcctctgttt tagtcgagat aagcccacag ctacgcggct agtcaaaatt cttctatcaa tctacctcct tactcctaag cttcactttc agaaatatac gagaagaaag caagaggaag accgtcaagt cctcatcgga tttcgagatc tg ttacgggctt acgaaggcca aggagtttct tgggaaacaa ctatttatct cacatttctc ggatttccga tatcagaccg aatcatgtct aacaaaactc atagtcttcg tttaacctca 1860 1920 1980 2040 2100 2160 2192 <210> 17 <211> 2658 <212> DNA <213> Arabidopsis thaliana <220> <221> promoter <222> (1)..(2658) <223> transcription regulating sequence from Arabidopsis thaliana gene At 2gO 1180 <400> 17 ataatcaacc gaaacacgat aagt tat aag agctgttgga aaagaccatt taattttgag agagagacgt gaaatgcaaa ataaaatatt tttggtaact ttgacattct taaaactata tgtgcaagtt tataaaaaac tttgtgattg caaaaccaaa ttttgcccta cacacatcat tgtttccaat aattccattt atatctggat tgagaaaagg gtcaatagaa aaatttcgtt agcaagttgc ttctcgtttg taaaaggaga aagaggtgac aagacaggcg ctacagaatt tttgtatatc aatgatgatg agtgaaacaa taaatcattt ttttttggag ct ctt tct ct tatttatatc ttttagctat tgttaaaaaa gtaaatgtca accttaaaaa tcaaattaaa taacaaaaaa ttttaagtcc ccacccatac tccagctttt tctaattgcg gcaagataat ttacaataaa catgcactct atcgtgattt tttcaagaaa ttgactacat taaagtttgt gacgtgttaa ttctctcttt tttgtgcatt ggaaacctaa taaaatttaa accgtgatgt ct at tgt cct cataaaatat taattttatc agctgagaat aactatttat ttaaagccgt attggtgtgt aaaaaatcac accaccaaac acaattatag aagtaggatt tgctccataa ttgaacgtcc caaagaaagg ccaaaataaa ggtttatgtt agcccttata tttatggttg gtaatgtata tgtgtatatc ttcttacgat accgagtgag aagagtaagc ttaatattaa cgtcaaaaca tctttttttt ataaaagaaa ggaatagtgc aaaatttttt actgttttaa tatgctattt tgatgagtgc acataaaagt tggacccact tagtccacct attattggtt acccttattc aagtcatcat ttccgagtcc ataaaaaaca tgtcgatctc ataagaactc ggaatgaaat aacagaatct tggaattagg caatatgcaa ataactaaat aaacaaacaa ttcgttttta ttcaagatgt cttcttttac tcttattagt aaaactgtta ataacacttt agtgttaagt ttttaataaa aaaagcgtag gtaattgtga a accc ct ac~t tttttctttt ggtaaaagat tagttcaacc catctttttt tcaagcatct gacaaagact atattataca tatgaaacct tcgaagatct cttttgtacg tattaaacat attagcaaat gatgtttcaa caaatcagca ctttaaggtt atattgatta tttttaactt aactaaaagt gaaaaaactg tggtaacaat ggttaccatt gaagacatga gcaaataaat ggaaaaaaga cagggtatac ctttctacac gcaaagaata ataattgtca tttctttctg ctcacgtcgg ttagacttgt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 PF 56134 gacttttcga tagtttccac atttgttgta ctggatatgt aaccccctat taaggcctaa accttcctaa gcgataactg ctctctgttt gcaagtttaa ttagatcaat tctcatctct ctcttattct ttttctttat aaaacattgt tctggatccg caatttgttc tgtcaagccc caattagaag gaggcgcaga cctcattatg tttcttcctc ttctaaaaag gtgtaatgga gtagtttgaa taagtaaggt aaagtgggac aaaagtcaaa tcttcttatt caacgcgtct tcctctgttt tagtcgagat ggcgcaattc cattttaaaa taatgaaaca tagagcaaag atccgataga taaccctcaa aaggtttagt gagggagg tacttactta aataagttcc ttatatattc tacatattag aactgaatca aagcccacag ctacgcggct agtcaaaatt cttctatcaa tctacctcct tactcctaag cttcactttc tcaggtaacc atcctaacat gtggtgtgct tgttgtcgtg atgaattgca gtagtatttc tgactgtgat cttcatcttc atttttggat tatttgctgg taaatagtta tctgacaaaa agaaatatac gagaagaaag caagaggaag accgtcaagt cctcatcgga tttcgagatc tgcatgacaa tcaatcgact tgaaaataaa tagcataacg tctttgacaa ttccgttgtt ttagagagcc gattgtttgt tatttggcac tctttcctat gctttagata agtccaaaaa cgagcaggga ttacgggctt acgaaggcca aggagtttct tgggaaacaa ctatttatct cacatttctc taqggtcgtt attctctgtt gtagttaaat aatcagacag agttctttgt actatcatcc attctttgtt gtttttggtg ccacaaacag ttttacacaa tgggctggct agaaattata aggcccataa ggatttccga tatcagaccg aatcatgtct aacaaaactc atagtcttcg tttaacctca tttctcttct ctactccctt agagtcaacg acaaaaagtg ttgcctaaac aaaccggaga tgtttagatc gcaggaccag 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2658 <210> <211> <212> <213> 18 1711 DNA Arabidopsis thaliana <220> <221> ODS <222> (1430) <223> coding for putative phosphatidic acid phosphatase <400> 18 gatccacatt caatagggtc actattctct aaagtagtta acgaatcaqa caaagttctt gttactatca gccattcttt tgtgtttttg tctctttaac gtttttctct gttctactcc aatagagtca cagacaaaaa tgtttqccta tccaaaccgg gtttgtttag gtggcaggac ctcatctcat tctctcttat cttttttctt acgaaaacat gtgtctggat aaccaatttg agatgtcaag atccaattag caggaggcgc ctcttagtcg tctggcgcaa tatcatttta tgttaatgaa ccgtagagca ttcatccgat ccctaaccct aagaaggttt agagagggag agatcttcac tttctgatga ttctcaggta acctcaatcg aaaatcctaa cattgaaaat acagtggtgt gcttagcata aagtgttgtc gtgtctttga agaatgaatt gcattccgtt caagtagtat ttcttagaga agttgactgt gatgattgtt g atg cag gag ata gat Met Gin Glu Ile Asp 120 180 240 300 360 420 480 536 ctt agt gtt cac act ata aag tcc cat gga gga aga gtc gct tct aaa 584 Leu Ser Val His Thr Ile Lys Ser His Gly Gly Arg Vai Ala Ser Lys PF 56134 cac aag cac is Lys His ggc ttg aac Gly Leu Asn gat Asp ot 0 Leu tgg T rp atc ata cto Ile Ile Leu gt c Val1 30 tao Tyr atc ttg att gc Ile Leu Ile Ala atc gag ata Ile Giu Ile aaa. gao atg Lys Asp Met atc tct cot Ile Ser Pro cgc tac gtg Arg Tyr Val atg act Met Thr gac ctc aag tac Asp Leu Lys Tyr oct Pro ttc aag gac aac Phe Lys Asp Asn gta cot atc tgg Val Pro Ile Trp tot Ser ttc Phe gtc Val cot gtg tac Pro Val Tyr gtg ott ott coo Val Leu Leu Pro ato Ile 80 ata gtg ttc gtc Ile Val Phe Val tgc Cys tao ctg aag agg Tyr Leu Lys Arg ctg oto ttc gc Leu Leu Phe Ala aca tgt gtg tao Thr Cys Val Tyr gat Asp 95 ggt Gly otg cac cac ago Leu His His Ser ato oto Ile Leu 100 ggg Gly gto ttg ata Val Leu Ile aag gta Lys Val ccc gao Pro Asp 135 ggc aag Gly Lys 105 aco Thr act Thr 110 oct Pro gcc Ala 120 ggc Gly gga ogo cot Gly Arg Pro gto ato act Val Ile Thr cgt Arg 125 gat Asp aao ttc tao Asn Phe Tyr tgg Trp 130 gtg Val Asp Ser Ile 115 ogo tgc tto Arg Cys Phe gta tgc cac Val Cys His aaa gag ctg Lys Glu Leu tat Tyr 140 aag Lys gog ttg gga Ala Leu Gly ggt Gly 145 ago Ser 632 680 728 776 824 872 920 968 1016 1064 1112 1160 1208 1256 1304 gca got gag Ala Ala Glu 150 cac His gto Val1 155 ttt Phe gaa ggo cac Giu Gly His aag Lys 160 ttc Phe tto cog ago Phe Pro Ser gga Gly 165 act tcc tgg Thr Ser Trp too Ser 170 aag Lys gog ggg ott Ala Gly Leu ott too ott Leu Ser Leu tao oto Tyr Leu 180 tot ggo aaa Ser Gly Lys tgc oto gtg Cys Leu Val 200 ogt gtg gat Arg Val Asp at c Ile 185 atc Ile goc tto aao Ala Phe Asn aat Asn 190 gco Al a gga oat gtg Gly His Val Ala Lys Leu 195 ggg ata tot Gly Ile Ser ttc cot ctg Phe Pro Leu ott Leu 205 ca 0 His got tgt ott Ala Cys Leu gt g Val 210 gao tao tgg Asp Tyr Trp tgg caa gat Trp Gin Asp gt 0 Val1 225 tto goa gga got Phe Ala Gly Ala 215 att Ile ggo aoc ott Gly Thr Leu gta Val 235 goc gco ttc tgo Ala Ala Phe Cys ogt cag tto tao Arg Gin Phe Tyr 000 Pro 245 gca Ala aao cot tao cao gaa gaa Asn Pro Tyr His Glu Glu gga tgg Gly Trp ggt ccc tao goo tat ttc aag Gly Pro Tyr Ala Tyr Phe Lys PF 56134 gct caa gaa Ala Gin Glu ttg agg gct Leu Arg Ala 280 cga Arg 265 atg Met 250 gga Gly gtc cct gtg Val Pro Val acc Thr 270 gat Asp tct ctg cag Ser Leu Gin atg Met 285 gaa tct ggc act tcc acc gct ccc aga Glu Ser Giy Thr Ser Thr Ala Pro Arg 295 300 attcattatt tggtttttca ttttgatttg gcc ctacatactg tatgtgtatt caaaactcta ctt cttcatgaaa ttgacgtttt tctggtgtcc gaq ccaatagact aatacggctc tagtgaacac ggt atcagttatt tcttaattcg t 255 260 tcc tcc caa aac gga gat gcc Ser Ser Gin Asn Gly Asp Ala 275 tca aca tct ctc gaa aac atg Ser Thr Ser Leu Glu Asn Met 290 tga tcctcctctc ttattatttg :gtcgtcg tgagattgtg aatggtgtag ;gtaccat tacatttttg taaatccact laggcttg ggtcgctcga ataatctcgt :atttaca tgtttgtgat ctaaactgaa 1400 1450 1352 1510 1570 1630 1690 1711 <210> <211> <212> <213> 19 302 PRT Arabidopsis thaliana <400> 19 Met Gin Glu Ile Asp Leu Ser Val His Thr Ile Lys Ser His Giy Gly 1 5 10 Arg Val Ala Ile Ala Ile Ser Lys His Lys His Asp 25 Trp Ile Ile Len Val Ile Len Tyr Arg Tyr Glu Ile Gly Len Leu Ile Ser Pro Phe Vai Gly Lys Asp Met Met Asp Len Lys Tyr Pro Phe Lys Asp Asn Thr Val Pro Ile Trp Ser 70 Vai Pro Val Tyr Val Len Len Pro Ile Ile Vai Phe Val Cys Phe Tyr Len Lys Len Giy Len Len Phe 105 Arg 90 Thr Cys Val Tyr Asp Len His His Ser Ala Val Leu Ile Thr Gly Val 110 Ile Thr Asp 115 Ser Ile Lys Val Ala Thr 120 Gly Arg Pro Arg 125 Pro Asn Phe I PF 56134 Tyr Trp 130 Arg Cys Phe Pro Asp 135 Gly Lys Glu Leu Tyr 140 Asp Ala Leu Gly Gly 145 Val Val Cys His Lys Ala Ala Glu Val 155 Lys Glu Gly His Ser Phe Pro Ser Gly 165 His Thr Ser Trp Phe Ala Gly Leu Thr Phe 175 Leu Ser Leu His Val Ala 195 Leu Ser Gly Lys Ile 185 Lys Ala Phe Asn Asn Glu Gly 190 Ala Ala Cys Lys Leu Cys Leu Ile Phe Pro Leu Leu 205 Leu Val 210 Gly Ile Ser Arg Val 215 Asp Asp Tyr Trp His Trp Gin Asp Val 225 Phe Ala Gly Ala Leu 230 Ile Gly Thr Leu Val 235 Ala Ala Phe Cys Arg Gln Phe Tyr Asn Pro Tyr His Glu 250 Glu Gly Trp Gly Pro Tyr 255 Ala Tyr Phe Gln Asn Gly 275 Lys 260 Ala Ala Gln Glu Gly Val Pro Val Thr Ser Ser 270 Asp Ser Thr Asp Ala Leu Arg Ala 280 Met Ser Leu Gln Ser Leu 290 Glu Asn Met Glu Gly Thr Ser Thr Ala Pro Arg 300 <210> <211> 2219 <212> DNA <213> Arabidopsis thaliana <220> <221> <222> <223> promoter (2219) transcription regulating sequence from Arabidopsis thaliana gene At3g45560 PF 56134 <400> attataactc atatgaggag gatactcctt gtagttttaa tgacgcttat gacaaaatca attattatga actacaattt atttttttat ttttcgcgat aaagaaccaa cttaaccggc aattgtaaaa atatagttgt tatctacgta tgtttatttg ttttatatga catcttaacc caatggtgaa gctaattcat tttgttgatg acttatttat tctacaaatt gattagttat tcatttttga tagagatatt taaacatatc ttggcgggac tattttatag tgtctccaca gtttaagttg agaaactgct tctattaatt acgaaaagct tgggcatggg tacaaaatat gcttctatat <210> 21 tatgttgcat ccaaatggat caacaactgt t t tt tggga cggaaactcg tatcctagtc taagacttga tttcttgcct caaaactaca caagttctgg atgtttgtgc cattcctgct gaatatatgt cataataata tcgctaatta aaattggatg ccactaagac attggtcgga acccaaatgg cat gtgt tt t gggaatatac agtggctgtc aaatgtttgc taaaataaaa tattgctttt tqatagcaat ataaataaaa gggtttggca atagatacaa gtatccgcgg taacacatca attttaacaa aggaatttta ctttcacaaa ttctaacgga atattatttg atatatggga cactggaaga gtcgacatgt cgtttattgt agaggattgg atttccatct aattttattt cgattataca aatcgagaat acgagtggat aacgcttcca gacggactga gattagttgc tatgcatatt ataatgggtt ccgaaagaat tcaatcccat tggttctagt catgcatgtt ttttctatgt atttttgcag actgatattt atttaactac cttccaaaat tttttatgac taattactac ttagtattaa ggacgttact tttgcaaact gttaaagtct ttggtcatg gttctgtcag tttttctagg attgcaacat taatgatagg gaactcacga ggaatcttca gtttgcactc gtttgcgtct tatcaacatt taacgatgaq ttgacataaa tgcttttgat aaaacgttac cgtacgtaat catcctctct gttagccgaa acaagagcag ctctaatatt cttctatcca cccaaacgat gtcttgatca agggttttct tgacgaaac caaacctctc cgaaagttca tcattttgtg aataattgtt cttcccaaaa atttaatatc tataacaatt aaaattacaa taaaataaat taataacaat tttatatata agtaaactaa tttcttatat acgttccagt tccttgtagg gatgaagata ttccctaaat acggcctaac tagacaactc attcaacaac ttagttttct tctatcggtg catgcaagtg aagatgaaaa gttttgtttt aacctaaaac accaacaaat tttcacttga caccgcaacg cacgaaatca ctttttacct aattttggta tccaaatttt tcaaatgctt tgatgggtaq gatccaattc caattgatta tattctgtat aaataaatcc tttgataatt tctctccgca atttaaacac cgattataag aatattaaga agattttttt tgtaaactat ctaactttaa aaacacaagc aaatattaag tcaacgtaga ccaaaaaaac ttcttataag tttagggatg cctaaagccc gcaatatata taatccgcca taccttttgg atcaacatcg aagctaggat ctttggttta tgatcgtcgt tttattataa catgttctgg ccattgatat ggaacacacc tcaactctga tactaattag ttttataaga gcaagtgata ctggtttttc ttaacaactc aatctgaaca ttgagtgaaa ttatatcttc tcaacttggg agaaagtaaa ttaattatac tacaaaatta aagcaaaccg caatgattca acgggacggg aaaacaaaaa gaaaataaat taaatagaaa tgtgcaaaaa tcaaataaag aagctccacc aacqaacaca ttctaaaata taaaattttq tctagactc tacatggttg cttggtagat gcgggtacga gttttgctac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2219 <211> <212> <213> 2218 DNA Arabidopsis thaliana <220> PF 56134 <221> <222> <223> 27 promoter (1)..(2218) transcription regulating sequence from Arabidopsis thaliana gene At 3g4 55 <400> 21 attataactc atatgtggag gatactcctt gtagttttaa tgacgcttat gacaaaatca attattatga actacaattt atttttttat ttttcgcgat aaagaaccaa cttaaccggc aattgtaaaa atatagttgt tatctacgta tgtttatttg tt ttat atga catcttaacc caatggtgaa gctaattcat tttgttgatg acttatttat tctacaaatt gattagttat tcatttttga tagagatatt taaacatatc ttggcgggac tattttatag tgtctccaca gtttaagttg agaaactgct tctattaatt acgaaaagct tgggcatggg tacaaaatat gcttctatat <210> 22 tatgttgcat ccaaatggat caacaactgt ttctttggga cggaaactcg tatcctagtc taagatttga tttcttgtct caaaactaca caagttctgg atgtttgtgc cattcctgct gaatatatgt cataataata tcgctaatta aaattggatg ccactaagac attggtcgga acccaaatgg catgtgtttt gggaatatac agtggctgtc aaatgtttgc taaaataaaa tattgctttt tgatagcaat ataaataaaa gggtttggca atagatacaa gtatccgcgg taacacatca attttaacaa aggaatttta ctttcacaaa ttctaacgga atattatttg atatatggga tacatggttg cactggaaga gtcgacatgt cgtttattgt agaggattgg atttccatct aattttattt cgattataca aatcgagaat acgagtggat aacgcttcca gacggactga gattagttgc tatgcatatt ataatgggtt ccgaaagaat tcaatcccat tggttctagt catgcatgtt ttttctatgt atttttgcag actgatattt atttaactac cttccaaaat tttttatgac taattactac ttagtattaa ggacgttact tttgcaaact gttaaagtct ttggtcatgg gttctgtcag tttttctagg attgcaacat taatgatagg gaactcacga ggaatcttca cttggtagat gtttgcactc gtttqcgtct tatcaacatt taacgatgag ttgacataaa tgcttttgat aaaacgttac cgtacgtaat catcctctct gttagccgaa acaagagcag ctctaatatt cttctatcca cccaaacgat gtcttgatca agggttttct tgacgaaacc caaacctctc cgaaagttca tcattttgtg aataattgtt cttcccaaaa atttaatatc tataacaatt aaaattacaa taaaataaat taataacaat tttatatata agtaaactaa tttcttatat acgttccagt tccttgtagg gatgaagata ttccctaaat acggcctaac tagacaactc gcgggtacga attcaacaac ttagttttct tctatcggtg catgcaagtg aagatgaaaa gttttgtttt aacctaaaac accaacaaat tttcacttga caccgcaacg cacgaaatca ctttttacct aattttggta tccaaatttt tcaaatgctt tgatgggtag gatccaattc caattgatta tattctgtat aaataaatcc tttgataatt tctctccgca atttaaacac cgattataag aatattaaga agattttttt tgtaaactat ctaactttaa aaacacaagc aaatattaag tcaacgtaga ccaaaaaaac ttcttataag tttagggatg cctaaagccc gcaatatata gttttgctac taatccgcca taccttttgg atcaacatcg aagctaggat ctttggttta tgatcgtcgt tttattataa catgttctgg ccattgatat ggaacacacc tcaactctga tactaattag ttttataaaa gcaagtgata ctggtttttc ttaacaactc aatctgaaca ttgagtgaaa ttatatcttc tcaacttggg agaaagtaaa ttaattatac tacaaaatta aagcaaaccg caatgattca acgggacggg aaaacaaaaa gaaaataaat taaatagaaa tgtgcaaaaa tcaaataaag aagctccacc aacgaacaca ttctaaaata taaaattttg tctagact 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2218 PF 56134 <211> <212> <213> 1512 DNA Arabidopsis thaliana <220> <221> CDS <222> (1)..(1512) <223> coding for zinc finger (C3HC4-type RING finger) family <400> 22 atg tat gac Met Tyr Asp 1 aag ggt ttt Lys Gly Phe gac caa gag Asp Gin Glu gac tct gcc Asp Ser Ala cta aaa Leu Lys 5 gtg age Val Ser ccc tca ccg ttc Pro Ser Pro Phe cac His 10 ggc Gly atg aat agg ctt tac ttc Met Asn Arg Leu Tyr Phe gag gaa act Glu Glu Thr gat Asp aag Lys 25 cat His ttt ggg gtt Phe Gly Val aag cta ctg Lys Leu Leu tat Tyr 40 gag Glu atc aag ggt Ile Lys Gly tca Ser gca Ala gct att tgc Ala Ile Cys cgt cat cat Arg His His ttg aag cga Leu Lys Arg ata aca gtt Ile Thr Val get gag ctt Ala Glu Leu gga Gly 65 tgc Cys cta Leu ace Thr cat His atc gaa get Ile Glu Ala gtg Val 70 att Ile ttg ggg atc Leu Gly Ile aac Asn atg Met atc tca ttc Ile Ser Phe gat cat gat Asp His Asp ttc gaa ttg Phe Glu Leu gtc Val gat Asp ggg ata tcg Gly Ile Ser gtc cca Val Pro gag caa gat Glu Gin Asp aac Asn 100 act Thr gct ttg ctt Ala Leu Leu gat gtg caa Asp Val Gin aaa caa ttt Lys Gin Phe 115 aag ttt gct Lys Phe Ala 130 tct age atc Ser Ser Ile cct Pro 120 atg Met ttg atg act Leu Met Thr cgt att aga Arg Ile Arg 110 aat caa get Asn Gin Ala gaa att agc Glu Ile Ser tat aag ctt Tyr Lys Leu gca Ala 135 cag Gin gaa aca ata Glu Thr Ile gtt Val 140 ggt Gly 288 336 384 432 480 528 576 ata Ile 145 gat Asp gat atg gcg cct Asp Met Ala Pro tct Ser 150 agg aag act Arg Lys Thr atc tgt ttc Ile Cys Phe gat ttc aaa Asp Phe Lys gct Ala 165 gag Glu gag cat atg ttt Glu His Met Phe tgc atg aca caa Cys Met Thr Gin tct Ser 170 tat Tyr gat tta tgt Asp Leu Cys ggc cat Gly His 175 cta ctc Leu Leu caa ttc tgt gtg Gin Phe Cys Val ata aaa gtg agg Ile Lys Val Arg PF 56134 gag gaa agt Glu Glu Ser 195 180 gag atg aga tgc Glu Met Arg Cys cct Pro 200 ctt Leu tat caa tgc Tyr Gin Cys gag Glu 205 cta Leu tcc aag tta Ser Lys Leu aga gag atg Arg Glu Met act gtt Thr Val 210 tgg gaa TrD Glu gta Val cga tgt gcc Arg Cys Ala ttg act ccg Leu Thr Pro gaa Glu 220 gtg Val cat agg agc His Arg Ser 225 tat Tyr caa Gin 230 tgt Cys gaa tcc gtt Glu Ser Val gca gac aaa Ala Asp Lys tgc caa atc Cys Gin Ile gct tgg ctt Ala Trp Leu tta Leu 250 att Ile caa atg gag Gin Met Glu gat ggt gct tta Asp Gly Ala Leu 260 tgt ggc ata aca Arg Gly Ile Thr gta gta agt Val Val Ser gca tca gct Ala Ser Ala gca Ala 270 gcg Ala ttc aga Phe Arg 255 aag ttc Lys Phe gat ata Asp Ile act tgt cgt Thr Cys Arg agt ttt Ser Phe 290 atc agc Ile Ser 275 gct Ala gcg Ala 280 atg Met aca cat gtg Thr His Val aat aca aga Asn Thr Arg gag Glu 295 ttg Leu aat gga agc Asn Gly Ser aag Lys 300 tta Leu gtt gcc ttc Val Ala Phe gaa agg aga Glu Arg Arg 305 agt Ser tgc Cys 310 acg Thr tgg agt atg gct Trp Ser Met Ala 315 ctt caa ggg Leu Gin Gly ttt Phe 320 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 gag cgg gga Glu Arg Gly aca Thr 325 tat Tyr gga ttt gcg Gly Phe Ala gca att tgt gac Ala Ile Cys Asp caa gag Gin Glu 335 aat aag cta Asn Lys Leu cat acc aag His Thr Lys ggt Gly 345 acg Thr tca ctt cat cat Ser Leu His His att aca att Ile Thr Ile 355 gaa gct gtg Glu Ala Val gag gct gag Glu Ala Glu tcc tta aaa Ser Leu Lys caa Gin 365 tac Tyr gac tcc acc Asp Ser Thr 350 gga cta acc Gly Leu Thr tgc gat cat Cys Asp His act Thr 385 att Ile 370 aaa Lys aga ttg ggg ata Arg Leu Gly Ile 375 ttc gat ttg gtc Phe Asp Leu Val tat atc aaa Tyr Ile Lys ctt Leu atg ggg aca Met Gly Thr tcc Ser 395 gcg ctt gag gat Ala Leu Giu Asp gcc ttg cta atg Ala Leu Leu Met gat gtg cat cgc atc cga aaa caa Asp Val His Arg Ile Arg Lys Gin ttg aag Leu Lys 415 ctt ata Leu Ile tct agc aat cct att ctg gag act aga act caa att agt atg Ser Ser Asn Pro Ile Leu Giu Thr Arg Thr Gin Ile Ser Met PF 56134 aac ttg caa Asn Leu Gin 435 ccg agc cgg Pro Ser Arg aaa cca act Lys Pro Thr 425 ctt Leu aat gga gtc Asn Gly Val atc Ile 445 430 aag cgc gcc Lys Arg Ala tac ata ccg Tyr Ile Pro 450 ttt Phe 455 gcg Ala gcc ccg aag cca Ala Pro Lys Pro ctc aaa cga cca Leu Lys Arg Pro ccg Pro 465 tct Ser ago Ser cgt gaa aaa Arg Glu Lys cac His 470 tecc Ser agg caa aga Arg Gin Arg ctt Leu 475 aaa acc att Lys Thr Ile 1344 1392 1440 1488 1512 cca agg atc att Pro Arg Ile Ile 485 ggt aaa ata Gly Lys Ile tct ttt atg gga Ser Phe Met Gly ctt tcg Leu Ser 495 gaa atg ata Glu Met Ile gag aat ctt tga Glu Asn Leu <210> 23 <211> 503 <212> PRT <213> Arabidopsis thaliana <400> 23 Met Tyr Asp Leu Lys Pro Ser Pro Phe 1 5 Met Asn Arg Leu Tyr Phe Lys Gly Phe Asp Gin Glu Val Ser Glu Glu Thr Lys 25 Gly Phe Gly Val Ala Ile Cys Arg His His Asp Lys Leu Leu Tyr His Ile Lys Gly Asp Ser Ala Ile Thr Val Glu Ala Glu Leu Thr Ala Leu Lys Arg Gly Leu Ile Glu Ala Val 70 Gly Leu Gly Ile Asn His Ile Ser Phe Cys Asp His Asp Ile Phe Glu Leu Val Met Gly Ile Ser Val Pro Glu Gin Asp Asn 100 Ile Ala Leu Leu Asp Asp Val Gin Arg Ile Arg 110 Lys Gin Phe Thr Ser Ser Ile Pro Val Leu Met Thr Arg Asn Gin Ala PF 56134 Lys Phe 130 Ala Tyr Lys Leu Al a 135 Met Glu Thr Ile Val1 140 Ser Glu Ile Ser Ile 145 Asp Met Ala Pro Gln Arg Lys Thr Gly Ile Cys Phe Asn 160 Asp Asp Phe Lys Al a 165 Glu His Met Phe Val Asp Leu Cys Gly His 175 Gln Phe Cys Glu Glu Ser 195 Val1 180 Glu Cys Met Thr Gln 185 Tyr Ile Lys Val Arg Leu Leu 190 Ser Lys Leu Glu Met Arg Cys His Tyr Gln Cys Glu 205 Thr Val 210 Val Arg Cys Ala As n 215 Leu Leu Thr Pro Glu 220 Leu Arg Glu Met T rp 225 Glu His Arg Ser Lys Glu Ser Val Val 235 Val Ala Asp Lys Tyr Cys Gln Ile Glu 245 Cys Ala Trp Leu Cys Gln Met Glu Phe Arg 255 Asp Gly Ala Arg Gly Ile 275 Leu 260 Asp Val Val Ser Leu 265 Ile Ala Ser Ala Ala Lys Phe 270 Ala Asp Ile Thr Thr Cys Arg Al a 280 Ser Asn Thr Arg Asp 285 Ser Phe 290 Ala Thr His Val Met Asn Gly Ser Glu Val Ala Phe Ser Glu Arg Arq Cys 310 Leu Trp Ser Met Leu Leu Gln Gly Phe 320 Ser Glu Arg Gly Thr 325 Thr Gly Phe Ala Val 330 Ala Ile Cys Asp Gln Glu 335 Asn Lys Leu Leu 340 Tyr His Thr Lys Gly 345 Ser Leu His His Asp Ser Thr 350 Ile Thr Ile Leu Glu Ala Glu Leu Thr Ser Leu Lys Gln Gly Leu Thr I PF 56134 360 Glu Ala 370 Val Arg Leu Gly Ile 375 Thr Tyr Ile Lys Ile 380 Tyr Cys Asp His Thr 385 Lys Leu Phe Asp Val Met Gly Thr Ala Leu Glu Asp Ile Ala Leu Leu Met 405 Asp Asp Val His Arg 410 Ile Arg Lys Gin Leu Lys 415 Ser Ser Asn Asn Leu Gin 435 Ile Leu Glu Thr Thr Gin Ile Ser Met Leu Ile 430 Lys Arg Ala Trp Lys Pro Thr Leu Asn Gly Val Pro Ser 450 Arg Tyr Ile Pro Phe 455 Ala Pro Lys Pro Pro 460 Leu Lys Arg Pro Pro 465 Ser Arg Glu Lys Ala Arg Gin Arg Leu 475 Val Lys Thr Ile His 480 Ser Pro Arg Ile Ile 485 Ser Gly Lys Ile Gly 490 Ser Phe Met Gly Leu Ser 495 Glu Met Ile Glu Asn Leu <210> <211> <212> 24 2042 DNA <213> Arabidopsis thaliana <220> <221> <222> <223> promoter (1)..(2042) transcription regulating sequence from Arabidopsis thaliana gene At4g00580 <400> 24 cttcttctta cgtttccagt gtgaattgga tttgtgttct cctcctcttc tccagtagaa gaagctgtgt ctgaggaaga gggaagatgt ctttaagaac agctgcccga agaatagtaa aagggtcaga caataaataa agctttccaa ttttctaaaa ttgtaaagaa gaaataattc ttttattcaa aagtcaataa atacaaggaa acaaaaactg aagttgcccg aaggaaaacg PF 56134 ggcaaagagg cattttccag tagatcaatg ctggtaacaa aaaggaaagg caaaaaaata ttttttttcc aaattaaaca tttgacataa ataactataa atccaaatag gtacgtagtc ttgactatgg gttttaataa gtaattctct actatgtatg tgtgacttat gctcaacaag tcaggtaact cttaaaccag ttctccctgC gatcaatgat acttgtcttt aatccatctg agctttgctt ataacgtgag ttcaatgagt ctacacgttg cttttcatat ttcattttct tg gtataaaagt gcatattggt acacgtgtat at at ta cgt c taatatataa ttcgctgcgg taccattcat tagttaaggg agaaaacatt tcttaattgt gtaaaaaaga tgaactctaa ttaattctat ataaatattt aatacactct actttgtttg cttcaacatc aacaatcttt tgtaactaca aaaactaaac aacttcattt tctacaccgg tgaaaattta ctccaaccca ttcaacaatt gcccactcat cataaaccaa tgttttccca tcctctacga tctcaacttc aaatgtggat gatgagacta ccgcgttctg ttcctaaaat atttgagatt cgacttgaca tcatgtcaag caagggaaaa caagatgtgt cttttgtcgg aactcgaata acaaaaatat ttttggaaat ggtgtttcta gatgtttgat ccttgtctac tcctcactgt cgaaccctgt aaacacacgt cgttttcttt tagctcatga cattacttgg tggttctttg ttgtcctgtg cctagtattc ttggcccata aagagagaag agtaattaag aagaaactcg atcaaaaccc atatagatcg gaggtcaaag gtgtttccgc aaaaaaagta cgagtgtttt ccgtcagctt atttcgaatc aaaaaagaat tggtatttca ccaataaact aaaattcata tagtattttt agagaaatct actccatcag cttagaacct tttctttgta tttgaatttc cttgatgtct agttgtcagt gcttgtctga agagaaaacc agctgtttct cttgagatag aaaagccctt ctggtttcct gagaaagaag acgaaaggac cgtttttgat cttataaata tattttgcat ccacgtggac gatattggct ttttacccat ccaagtagag aagtgtaaac ttaactctta ctataaattt gcatgcattt taatcaaata cgatccaaaa tacgatatga cttttacatt atatatgtat caacttcttc gttcaagtac ccttaaacac ttaaaccttg atacgactgc ttctcaattt ggcacccatt ataaacttca tcaatgattc acttgcatct agctaacttc tagagaatct ttatgatctg gatgaaaaga ttcccaaaaa tcaaacagag ttctcttaca ggttgtgaat aactcaccgt gcgtgtgcct aaattagaga cattctttta aacagtgctt gatcacagca atttatttat ctactaacaa ttctttgtcg aaaatattac gagaaagatc ggttcttagt acaggcccag atgaaataga tgaagacatc cagcaatgca aaaaaaccgc cccacttgag gatcccgagc cttcacttta tacaccaggt ctcttgataa ctcgccttat ctgtcttcat gtcaaaccct gagaatcgac aaacat aaag agagtggagt ataatcttac 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2042 <210> <211> <212> <213> <220> <221> <222> <223> 2044 DNA Arabidopsis thaliana promoter (2044) transcription regulating sequence from Arabidopsis thaliana gene At 4gO 0580 <400> cttcttctta cgtttccagt gtgaattgga tttgtgttct cctcctcttc tccagtagaa PF 56134 gaagctgtgt aagggtcaga ttttattcaa ggcaaagag cattttccag tagatcaatg c tggtaacaa aaaggaaagg caaaaaaata caaaattaaa attttgacat aaataactat cgatccaaat acgtacgtag tcttgactat gtgttttaat aggtaattct qaactatgta tctgtgactt cagctcaaca gctcaggtaa agcttaaacc gcttctccct tagatcaatg gtacttgtct aaaatccatc atagctttgc atataacgtg ctttcaatga acctacacgt agcttttcat gtttcatttt actg ctgaggaaga caataaataa aagtcaataa gtataaaagt gcatattggt acacgtgtat atattacgtc taatatataa ttcgctgcgg cctaccattc catagttaag aaagaaaaca aatcttaatt aggtaaaaaa tctgaactct ggttaattct aaataaatat ctaatacact tgactttgtt atcttcaaca agaacaatct cttgtaacta agaaaactaa gcaacttcat attctacacc tttgaaaatt tgctccaacc ttttcaacaa aggcccactt gtcataaacc tgtgttttcc attcctctac cttctcaact gggaagatgt agctttccaa atacaaggaa aaatgtggat gatgagacta ccgcgttctg ttcctaaaat atttgagatt cgacttgaca attcatgtca ggcaagggaa ttcaagatgt gtcttttgtc gaaactcgaa aaacaaaaat atttttggaa ttggtgtttc ctgatgtttg tgccttgtct tctcctcact ttcgaaccct caaaacacac accgttttct tttagctcat ggcattactt tatggttctt cattgtcctg t tcct agt at atttggccca aaaagagaga caagtaatta gaaagaaact tcatcaaaac ctttaagaac ttttctaaaa acaaaaactg atatagatcg gaggtcaaag gtgtttccgc aaaaaaagta cgagtgtttt ccgtcagctt agatttcgaa aaaaaaaaga gttggtattt ggccaataaa taaaaattca attagtattt atagagaaat taactccatc atcttaqaac actttctttg gttttgaatt gtcttgatgt gt agt tgt ca ttgcttgtct gaagagaaaa ggagctgttt tgcttqagat tqaaaagccc tcctggtttc tagagaaaga agacgaaagg agcgtttttg cgcttataaa cctattttgc agctgcccga ttgtaaagaa aagttgcccg ccacgtggac gatattggct ttttacccat ccaagtagag aagtgtaaac ttaactctta tcctataaat atgcatgcat cataatcaaa ctcgatccaa tatacgatat ttcttttaca ctatatatgt agcaacttct ctgttcaagt taccttaaac tcttaaacct ctatacgact gtttctcaat gaggcaccca ccataaactt cttcaatgat agacttgcat ttagctaact cttagagaat agttatgatc acgatgaaaa atttcccaaa tatcaaacag atttctctta agaatagtaa gaaataattc aaggaaaacg ggttgtgaat aactcaccgt gcgtgtgcct aaattagaga cattctttta aacagtgctt ttgatcacag ttatttattt tactactaac aat tctt tgt gaaaaatatt ttgagaaaga atggttctta tcacaggccc acatgaaata attgaagaca tgcagcaatg gcaaaaaacc ttcccacttg ttgatcccga cacttcactt tctacaccag ctct ct tga t tcctcgcctt ctctgtcttc tggtcaaacc gagagaatcg aaaaacataa agagagtgga caataatctt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2044 <210> <211> <212> 26 954 DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (954) <223> coding for COPl-interacting protein-related protein PF 56134 I <400> 26 atg gct gat tec ggt tcg caa tgt gtc ttt gtg aag act agt att gat 48 Met Ala Asp Ser Gly Ser Gin Cys Val Phe Val Lys Thr Ser Ile Asp 0 1 5 10 ace cgt ttg ggg ota ctc ctt gac agt cat gat age gtg tot tct tto 96 Thr Arg Leu Gly Leu Leu Leu Asp Ser His Asp Ser Val Ser Ser Phe 25 aaa gac aaa ttc tgc aaa gaa cac gaa ctg tgt ttt cca agt gtt ggc 144 Lys Asp Lys Phe Cys Lys Glu His Glu Leu Cys Phe Pro Ser Val Gly 35 40 aac atc act gtt tct gcc ttg aag gtt aac gtg agt ggt aat gat tat 192 Asn Ile Thr Val Ser Ala Leu Lys Val Asn Val Ser Gly Asn Asp Tyr r 50 55 cac ttg tcc gat tct atg ata ttg aaa aaa get ctt caa ggc ctc agt 240 S 15 His Leu Ser Asp Ser Met Ile Leu Lys Lys Ala Leu Gin Gly Leu Ser 70 75 aac gag gac ttt ttt cta tec gtt gac ctc gta cgt gtc cag gag aaa 288 Asn Glu Asp Phe Phe Leu Ser Val Asp Leu Val Arg Val Gin Glu Lys 90 agt gag ctg cag att ggt gaa gca gtt gag aaa aaa acg agg aag aga 336 Ser Glu Leu Gin Ile Gly Glu Ala Val Glu Lys Lys Thr Arg Lys Arg 100 105 110 aaa tcg aaa agt gcc aac aat agt aga aag aaa ctc tcc ata gag acg 384 Lys Ser Lys Ser Ala Asn Asn Ser Arg Lys Lys Leu Ser Ile Glu Thr 115 120 125 cca acg gaa gca aaa ggc ctt gaa agt ggt gag gga act gtc act agg 432 Pro Thr Glu Ala Lys Gly Leu Glu Ser Gly Glu Gly Thr Val Thr Arg 130 135 140 ttg gaa gag aat cag aat att tgt gat gta gat caa gag gaa cct gtc 480 Leu Glu Glu Asn Gin Asn Ile Cys Asp Val Asp Gin Glu Glu Pro Val 145 150 155 160 gat ggt cat acc ata gat gtt gag gcc aag att gat ctt tea ggg aca 528 Asp Gly His Thr Ile Asp Val Glu Ala Lys Ile Asp Leu Ser Gly Thr 165 170 175 ate gaa caa gac gac gtt caa aag gag gta gca aat get gat tta aac 576 Ile Glu Gin Asp Asp Val Gin Lys Glu Val Ala Asn Ala Asp Leu Asn 180 185 190 atg att gac caa gac aag gat ctt gaa aat gat aat ctt cta gct gaa 624 Met Ile Asp Gin Asp Lys Asp Leu Glu Asn Asp Asn Leu Leu Ala Glu 195 200 205 tta aac caa act agt gat gat gct gag aaa gaa ggg atc ata ggt ctt 672 Leu Asn Gin Thr Ser Asp Asp Ala Glu Lys Glu Gly Ile Ile Gly Leu 210 215 220 gtt aat gct act tct gaa gct att gaa aac gaa act gag atg agt gtc 720 Val Asn Ala Thr Ser Glu Ala Ile Glu Asn Glu Thr Glu Met Ser Val PF 56134 225 aag Lys gaa aaa gat Glu Lys Asp 230 gat Asp gag gag gct Glu Glu Ala aaa Lys 250 ccg Pro 235 tct Ser gag aag cct Glu Lys Pro aaa aag Lys Lys 255 aaa aat aga Lys Asn Arg gca Al a 260 aaa gtc aag Lys Val Lys act Thr 265 gag Glu act aaa gaa Thr Lys Glu gtt gct tct Val Ala Ser 275 ccg cag gag Pro Gln Glu agc tca agg aac Ser Ser Arg Asn gct Ala 280 gtt Val gaa gat ggt Glu Asp Gly gt c Val1 285 gat ggt cta Asp Gly Leu 270 tcg aga gat Ser Arg Asp aag aga tcg Lys Arg Ser taa aat gtg gct Asn Val Ala gtg aag aag Val Lys Lys 290 aag Lys ccg aac Pro Asn 300 gat gct Asp Ala aag Lys 305 gaa cag tct Glu Gln Ser tca Ser 310 att gtt gag Ile Val Glu <210> 27 <211> 317 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Ala 1 Asp Ser Gly 5 Ser Gln Cys Val Phe 10 Val Lys Thr Ser Ile Asp Thr Arg Leu Lys Asp Lys Gly Leu Leu Leu Asp Ser 25 His Asp Ser Val Ser Ser Phe Ser Val Gly Phe Cys Lys Glu His Glu Leu Cys Phe Asn Ile Thr Val Ser Ala Leu 55 Lys Val Asn Val Ser Gly Asn Asp Tyr Leu Ser Asp Ser Met Ile Leu Lys Lys Leu Gln Gly Leu Ser Asn Glu Asp Phe Leu Ser Val Asp Leu Val Arg Val Gln Glu Lys Ser Glu Leu Ile Gly Glu Ala Glu Lys Lys Thr Arg Lys Arg 110 Lys Ser Lys Ser Ala Asn Asn Ser Arg Lys Lys Leu Ser Ile Giu Thr PF 56134 115 Pro Thr 130 Glu Ala Lys Gly Leu 135 Glu Ser Gly Glu Gly 140 Thr Val Thr Arg Leu 145 Glu Glu Asn Gin Ile Cys Asp Val Gin Glu Glu Pro Asp Gly His Thr Asp Val Glu Ala Ile Asp Leu Ser Gly Thr 175 Ile Glu Gin Met Ile Asp 195 Asp 180 Asp Val Gin Lys Glu 185 Val Ala Asn Ala Asp Leu Asn 190 Leu Ala Glu Gin Asp Lys Asp Glu Asn Asp Asn Leu 205 Leu Asn 210 Gin Thr Ser Asp Asp 215 Ala Glu Lys Glu Gly 220 Ile Ile Gly Leu Asn Ala Thr Ser Glu 230 Ala Ile Glu Asn Glu 235 Thr Glu Met Ser Lys Glu Lys Asp Asp Glu Glu Ala Ser Glu Lys Pro Lys Lys 255 Lys Asn Arg Val Ala Ser 275 Ala 260 Lys Lys Val Lys Pro Thr Lys Glu Asp Gly Leu 270 Ser Arg Asp Ser Ser Arg Asn Ala 280 Glu Glu Asp Gly Pro Gin 290 Glu Asn Val Ala Lys 295 Val Val Lys Lys Pro 300 Asn Lys Arg Ser Lys 305 Lys Glu Gin Ser Asn Ile Val Glu Glu Asp Ala 315 <210> <211> <212> 28 2092 DNA <213> Arabidopsis thaliana <220> <221> promoter PF 56134 <222> <223> (2092) transcription regulating sequence from Arabidopsis thaliana gene At).g 54480 <400> 28 ttttgacgaa dgccttttta aaagatgaag aactctttag agtaaagact gaatgagact agacgtctac taacaattaa taatttattt attaatggtt atttggaaaa aatcaataat agcttgaagg atgtgtttta ttcaccatgg tgtagtaagc atattgccac tgagagtact ctttgaaacc tcaagctttc gtgtttttgc atcatcaaaa ctgtagaact ccttaatctc tagtttcacg aaatcttatc tagtggatat aaccaattat aaaatgaaaa gaaaatagtg attctttcac caaccataaa aaacaatagc tgttgaattc gagtttaatt tatacaaata t tggcgt gt c aaaaactaaa ttaagagtct accgagttgg agctagtttt taaagatttt tcaagtccaa agtccaaatt ttcaagcata aagtatttaa cattagaaga ctttggtgaa ataaataaat tccatgtcaa cgacacattc aataaatcaa tgcagcctaa ttcaatataa agacaaaaac gatcagccct gctgctatac caagctatct gagcgcatgt gctagacttg tataatgttt tttctcaatc gcaatccgca tgtaaaatcg tgtatcactg ttgaattgac catatcattc aatcacttac ttataaccat cgtttctttt gctttattta cattgtgaaa ttaatcttaa cgagttacct gcgtttgttg tacgatcaat ctgatttaca cgataatatt ttttgccatt aacgaagcag attccttatc aacacatcaa gcaaacagag atccaaaaca cacagtttta tgaatatatc tcatccaaaa agaacatcaa aaaatgaaga agtcaacaag aaggtccaac ttagcagcca ggaatcggca aaaacaggtt caattctaag aagatttaat tttgcttgat ataaaaaaca tatccttttc ttgacttact gatgatgggt atgtgccaca ggatgtattt tttttggacg gaactttttg atgatatgaa aaagacttgg agtt t tagaa atgctctcat tggaatggtt ataattcacg ttcaatatca tgtggtttaa ctgaagaatt aacgaattgg tcttacttgt acttttcata attacataac gacaataact gtaaatatca gcaacttgca tgattataga ctaattttgt aagagatcaa aatggagaac ttgtggctgg gtggaggttt caagcttcta tggtaacatg tttatgcatt agatttgatt taccgtagaa tatatttgaa tattacgtgg gttttctacg aaaacttctg gcattgaact aatagaatgt actttttgat agcacttaat aagagttgac aaaaaaacat aatcagtttt tgcaaaatta attaattagt aaaaatatac aagtttttgt ttcaagatca aagatttgat cccttacaac aggatcacat agtttgttct gtttcttcag ttgtttagta aatgaaatgt acaagcaaac tattctactt tcaacataat atctttgaaa agtgttatac attcattcca tagtatacga cacacgtttt ttattgacta aaattatatt aatttgcgca tgtcccaaac tatatacata atctacactt actcattcac cacactctca caaatagatt atatttagag agttatccaa gttggttgat gtataaactt tctttctttg tccatataac gttgttggga aaattcatat tttacagatt cgtatgagaa gtcattgcaa agatagaaag tacggaatca ccttatcttt cttcttatgt cgtaagtcaa gctccccact aaaaaaagtt cttctcctct ccagggatat gaatcaaatt ttccttgctg atcacgactc aatagatgtt agaatctata atcttttaaa taattaaaat catccaataa tgaagtttgt tatgagtctt ggccta tcat atacatatct aatttctttg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2092 catttgcttc tctttttctt tcttttttaa atgttatttg ca <210> 29 <211> <212> <213> 1653 DNA Arabidopsis thaliana PF 56134 <220> <221> <222> <223> CDS (1653) coding for leucine-rich repeat family protein <400> 29 atg Met 1 aat Asn aat Asn cca Pro tat Tyr ttt Phe ttt Phe qat Asp aac Asn gat Asp 145 ata Ile att Ile cct Pro gat Asp ctg Leu tct Ser aat Asn tca Ser ctt Leu agc Ser act Thr 130 att Ile tca Ser ggc Gly gct Ala att Ile tta Leu tct Ser aac Asn cta Leu cca Pro aac Asn 115 acg Thr cca Pro aac Asn ttt Phe aca Thr agt Ser cga Arg atg Met ttc Phe aaa Lys aga Arg 100 tca Ser tta Leu agc Ser aat Asn ctt Leu 180 ata gtg Ile Vai 5 gga ctg Gly Leu atg aac Met Asn ggt gag Gly Glu tcc ggg Ser Gly 70 cac ttg His Leu 85 gaa aca Glu Thr ttc act Phe Thr tca gtt Ser Val tgg atg Trp Met 150 ttt tta Phe Leu 165 tct ctc Ser Leu cat gag His Glu ctc cct Leu Pro ggc tca Gly Ser 40 atg gta Met Val 55 aag cta Lys Leu aag ctc Lys Leu agc ttt Ser Phe ggg aag Gly Lys 120 ctt gac Leu Asp 135 tct aac Ser Asn gaa ggt Glu Gly atc gac Ile Asp ttg cag Leu Gin 10 gat aat Asp Asn 25 aga aat Arg Asn aac att Asn Ile cct aga Pro Arg tcc cac Ser His 90 act tcc Thr Ser 105 atc gga Ile Gly atg tcc Met Ser tta tct Leu Ser acg ata Thr Ile 170 cta tcg Leu Ser 185 ttc ttg Phe Leu atc ggt Ile Gly ggg ttt Gly Phe aca tcc Thr Ser cgc ttt Arg Phe 75 aac aat Asn Asn ctg gaa Leu Glu gtt ggt Val Gly aac aat Asn Asn 140 ggt tta Gly Leu 155 cca cca Pro Pro gga aac Gly Asn gat ttt Asp Phe tat gcg Tyr Ala caa ggg Gin Gly ctt gat Leu Asp gtc act Vai Thr ttt agt Phe Ser gag ttg Glu Leu 110 ttg ctt Leu Leu 125 ttt cta Phe Leu act atc Thr Ile tct ttg Ser Leu tta tta Leu Leu 190 tca gtg Ser Vai ctt ccg Leu Pro cat tta His Leu cta tct Leu Ser ggt tgt Gly Cys ggc cat Gly His aga gtt Arg Val agc tcc Ser Ser acg ggt Thr Gly ttg tcg Leu Ser 160 cta gcc Leu Ala 175 tct gga Ser Gly 48 96 144 192 240 288 336 384 432 480 528 576 624 tcc tta ccg tca cgt gtt ggt ggg gag ttt ggg ata aaa ttg ttc cta Ser Leu Pro Ser Arg Vai Giy Giy Giu Phe Gly Ile Lys Leu Phe Leu PF 56134 cac gac His Asp 210 atg etc acg Met Leu Thr gte Val 225 caa Gin caa Gin ata ctt gat Ile Leu Asp tta Leu 230 gag Glu 200 ggg ccg att cca gae Gly Pro Ile Pro Asp 215 egg tae aat eaa ett Arg Tyr Asn Gin Leu 235 age ata tat att ett Ser Ile Tyr Ile Leu acg Thr 220 tct Ser 205 ttg ttg gaa aag Leu Leu Giu Lys ggg agt att Gly Ser Ile cca Pro 240 672 720 768 ttt gte aat Phe Val Asn ace Thr 245 tct Ser tta atg aag gga aae Leu Met Lys Giy Asn 255 gat ttg aga aat ate Asp Leu Ara Asn Ile aae tta aca Asn Leu Thr atg tea agg Met Ser Arg eag Gin 265 tgt Cys aga ett Arg Leu tgt eta Cys Leu 290 ggt aca Glv Thr tta Leu 275 tat Tyr gat ett tea gat Asp Leu Ser Asp aae Asn 280 gga Gly aag ete aat gge Lys Leu Asn Gly tte Phe 285 aat Asn 270 ata eet tea Ile Pro Ser tea tat gta Ser Tyr Vai aat tta tea Asn Leu Ser ttt Phe 295 cca gag gat Pro Giu Asp gee ata acg Ala Ile Thr 305 ttt Phe aag Lys 310 ttt Phe att aet ccg tte Ile Thr Pro Phe aag Lys 315 tct Ser ttt tae gaa tee Phe Tyr Giu Ser gtg gta gag Val Val Glu gat Asp 325 tea Ser gtg gta ata Val Val Ile tee Ser 330 tat Tyr agt ttt eaa Ser Phe Gin gaa att Glu Ile 335 gaa ate aaa Glu Ile Lys act gaa tte Thr Giu Phe 355 tea age aat Ser Ser Asn ttt Phe 340 aae Asn atg aag ega Met Lys Arg agg Arg 345 gat Asp gat tet tat Asp Ser Tyr aat gae gta Asn Asp Vai ctt Leu 360 gtt Val tat atg tat Tyr Met Tyr gga Gly 365 ett Leu ttt gga gca Phe Gly Ala 350 atg gac cta Met Asp Leu gga agt ete Gly Ser Leu 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 gag tta agt Glu Leu Ser tea Ser 385 ata Ile 370 aag Lys ggg Gly 375 aat Asn ate cca gca Ile Pro Ala gag Glu 380 eta ega gte Leu Arg Val atg Met 390 tee Ser tta tet tge Leu Ser Cys tte ttg tee agt Phe Leu Ser Ser tea Ser 400 cca tet age Pro Ser Ser aat etc aag Asn Leu Lys gat Asp 410 ect Pro att gag age ctt Ile Giu Ser Leu tcg eat aae Ser His Asn tet tct ett Ser Ser Leu atg Met 420 gtt Val eaa gga agt Gin Gly Ser eaa eaa eta Gin Gin Leu ace Thr 430 tee Ser gac ctt Asp Leu 415 aac ctt Asn Leu gga ate Gly Ile gte ttt gat gtg Vai Phe Asp Vai tae aat aat tta Tyr Asn Asn Leu PF 56134 att ccc Ile Pro 450 gga aat Giv Asn caa gga agg cag Gin Gly Arg Gin ttt Phe 455 gga Giy aat acc ttt gac Asn Thr Phe Asp aaa agc tac ttg Lys Ser Tyr Leu cct ctt ctt Pro Leu Leu oca ccg acc Pro Pro Thr 465 aag Lys aat Asn 475 gga Gi y agt tgt gat Ser Cys Asp got Aila 480 aag aco tca Lys Thr Ser gat Asp 485 gat Asp gaa tca gaa aat Giu Ser Giu Asn gaa gaa gaa Giu Giu Giu gat gat Asp Asp 495 1392 1440 1488 1536 1584 1632 gaa got cot Giu Aia Pro tat gta act Tyr Vai Thr 515 oct ttg cgt Pro Leu Arg gtt Vai 500 aca Thr atg ttg gcc Met Leu Ala tat ttt agt agt Tyr Phe Ser Ser ttg ata ggc Leu Ile Giy att Ile 520 cgc Arg ata ott atg Ile Leu Met tgc Cys 525 too Ser got tog aot Aia Ser Thr 510 ttt gat tgt Phe Asp Cys ato goo toa Ile Ala Ser oga goa tgg Arg Aia Trp att gtc gat Ile Val Asp gtc Vai 545 530 aaa Lys agt atg ttg Ser Met Leu oct Pro 550 1653 <210> <211> <212> <213> 550 PRT Arabidopsis thaiiana <400> Met Pro Aia Thr Ile 1 5 Vai His Giu Leu Gin 10 Phe Leu Asp Phe Ser Vai Asn Asp Ile Asn Leu Leu Ser Giy Leu Leu Pro Asn Ile Giy Tyr Aia Leu Pro Giy His Leu Arg Met Asn Giy Ser 40 Arg Asn Gly Phe Gin Pro Ser Ser Met Gly Giu Met 55 Vai Asn Ile Thr Ser Leu Asp Leu Ser Tyr Asn Asn Phe Ser Giy 70 Lys Leu Pro Arg Arg Phe Vai Thr Giy Phe Ser Leu Lys His Leu Lys Leu Ser His Asn Asn Phe Ser Giy His PF 56134 Phe Leu Pro Asp Ser Asn 115 Arg 100 Giu Thr Ser Phe Ser Leu Giu Giu Leu Arg Vai 110 Leu Ser Ser Ser Phe Thr Gly Ile Giy Vai Gly Leu 125 Asn Thr 130 Thr Leu Ser Val Leu 135 Asp Met Ser Asn Phe Leu Thr Gly Asp 145 Ile Pro Ser Trp Ser Asn Leu Ser Giy 155 Leu Thr Ile Leu Ser 160 Ile Ser Asn Asn Phe 165 Leu Giu Gly Thr Pro Pro Ser Leu Leu Ala 175 Ile Gly Phe Ser Leu Pro 195 Leu 180 Ser Leu Ile Asp Leu 185 Ser Gly Asn Leu Leu Ser Gly 190 Leu Phe Leu Ser Arg Val Gly Gly 200 Giu Phe.Gly Ile Lys 205 His Asp 210 Asn Met Leu Thr Gly 215 Pro Ile Pro Asp Thr Leu Leu Giu Lys 220 Ser Gly Ser Ile Pro Val 225 Gin Ile Leu Asp Arg Tyr Asn Gin Leu 235 Gin Phe Val Asn Thr 245 Giu Ser Ile Tyr Leu Leu Met Lys Gly Asn 255 Asn Leu Thr Arg Leu Leu 275 Gly 260 Ser Met Ser Arg Gin 265 Leu Cys Asp Leu Arg Asn Ile 270 Ile Pro Ser Asp Leu Ser Asp Asn 280 Lys Leu Asn Gly Phe 285 Cys Leu 290 Tyr Asn Leu Ser Phe 295 Giy Pro Giu Asp Thr 300 Asn Ser Tyr Val Giy 305 Thr Ala Ile Thr Lys 310 Ile Thr Pro Phe Lys 315 Phe Tyr Giu Ser Phe Vai Val Giu Asp Phe Val ValliSeSeSrPhGnGi le Ile Ser Ser Ser Phe Gln Glu Ile P F 56134 325 Ser Glu Ile Lys Thr Glu Phe 355 Ser Ser Asn Met Lys Arg Arg 345 Asp Asp Ser Tyr Asn Asp Val Tyr Met Tyr Gly 365 Leu Phe Giy Ala 350 Met Asp Leu Giy Ser Leu Giu Leu Ser 370 Giy 375 Asn Ile Pro Ala Giu 380 Phe Ser 385 Ile ,Lys,Leu Arg Vai Met 390 Ser Leu Ser Cys Leu Ser Ser Pro Ser Ser Asn Leu Lys Asp 410 Pro Giu Ser Leu Asp Leu 415 Ser His Asn Ser Ser Leu 435 Ile Pro Gin 450 Met 420 Val1 Gin Giy Ser Gin Gin Leu Vai Phe Asp Vai 440 Asn Tyr Asn Asn Leu 445 Lys Thr Asn Leu 430 Ser Giy Ile Ser Tyr Leu Giy Arg Gin Thr Phe Asp Giu 460 Arg Gly 465 Lys Asn Pro Leu Leu Cys 470 Glu Pro Pro Thr As n 475 Giy Ser Cys Asp Lys Thr Ser Asp 485 Asp Ser Giu Asn Gly 490 Tyr Giu Giu Giu Asp Asp 495 Giu Aia Pro Tyr Vai Thr 515 Pro Leu Arg 530 Met Leu Ala Phe 505 Phe Phe Ser Ser Aia Ser Thr 510 Phe Asp Cys Leu Ile Giy Ile Leu Met Cys 525 Arg Ala Trp Leu Arg Ile Val Asp Aia Ser Ile Ala Ser 535 540 Vai Lys 545 <210> 3 Ser Met Leu PF 56134 <211> 2512 <212> DNA <213> Arabidopsis thaliana <220> <221> <222> <223> promoter (2512) transcription regulating sequence from Arabidopsis thaliana gene At 4 g114 <400> 31 ttcgccttaa aatttgttga tatttcggag actgcataac ttattctagt ctcttttcta aacctattta gataaaacat gaaaagaata gaatgtattt ctagtaagaa ccttcacacc cagagccgat cctaggttgt tactaacaat aaacgtttaa aagcattatc tgaacqtgaa ctaagaaaaa gggtaatttg tcgtttgatc ccacgatcaa tgtgaaaaca ttagcaatga tttacaccct cataatttct cagttgtagg agggagcttt ttatagaaga aaaaaaataa gggaatcaca ttcaattgtt tagcaaacac actctttcag ttcctaccaa tgtactaaac gtataaatgt ccaaccgggg aaacgacgag aaatttctta tgttaccttg ttccattcca gcttcttcaa ttcttcgagt tcagattgta ccaggggcgg attataagta ttagcttgta gttttcaata ttcgtccatc tttttgactt tgctcatttt tgatttatga acatccactt gtatatgtgt attgatatat gaaaatgaat aacaaagtta aaattgtggg aaacaaatta tttaataacg aatagtatat aatagtgccc cagattaaat ctgatggagt tgtcttaatt tcttgatgct tcaagtagcc aacaatgtga tcgtctttgg tatatatata agaagatttt acagcaaatg aaaaaaaaac ggggcttaaa tattaataat acaaaacaaa cagcaaaaaa ggatgggtcg attgtcacta tgttttatgc ggtaaagggt ttgagggcct tctccttgct tgcgacagcc cggttatata ttcacgattc tggaagttag atcagttctc gggaggatgc atctaggtgt aacttatgca cttacccagg ttcccccaat ttaactttct ttctaaaatg tatgaacaca cagaatttat ataataattt atttttctag agacatatgc tgtatttttt aattcaaaaa taataataaa ataatacatt tatatattta aaagagtaga aaagtaacat tggtacgaac acatttttag ctataaaaca cactatagaa aagaaggaac qgcctgccaa gtccaaacag atccaactct tttgttgtaa att t tgt aaa ccgttgagtt tcatgagctt atctgattat taccgttgtt ggtttcctgg gtagagcttc gt aat ccat g atgggatgtg gtttaaatga ttcgagacta tttatcaatt tttaaaattt tttgatatat atttcatagt tgaactacat tgataaaaaa atccgccact caatggaaat tttccgtggg aataccatac tttatttatt atgtatttac acataattac caaactgatt tatgtggtct tgaaagggaa acgtgaaaag gaagtaagta tcaacaatag attacaactt atggtttagc tcttaaaact gaaaaatttt accgcactaa gaagttgttt gtataa.ccaa ccgcctaatt ccgttgattg taatgtcacg tgccaatagc gggcacgtgc ttcagcacaa gttgaagtaa taatcactta ctttattaat ctattcattt ttttttttgg attttatcac taatatataa ggattgtacc gtaatgctgt ggttcgaaac aaatcaaatg at ttt t tct t tatttttaaa gacaaaaaaa atacaaaaag taccaatcaa ttccaaaata aaaatactat aaaaaaattg gaagttgtgg aaaaatatat ttcaaagttc atatgtggtc tggggcctat ctaaaacttt tgctcactac agcaaatcgg accggttatt ctgatttgtt gttcgttccc gctcgtgatg agagtcgtgc cctacattaa ctggttaagt cttttttctt tttcttcctt tgattagata acttttatgt gtacctatat aaacttatag taagtaaatt atggaagcgt ccgacaatcg ttggattcag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 PF 56134 tcaggtcatt t ggtt t tgt a atattactaa tgcttcgttg gtgcatctca catcagctca aatgccttgg atatgaagtg ctttaatctt t tact gtct g tttgggtctt tgttacgttg catcattaac ttgctttgat atattttaag tacggatatg tactttaaac tgctgagaaa tggattgctc aggagattca atttttgaaa tggttctctg tttgacaact tacatactaa ccattgtcaa gtaataagat aatttgttgg ttcaacaagc cttcaacatt attttgtcta tgctgagaga ccccaaacac caatgcctct aagctattga ttatttaact atagtgggcg cttttaataa tgctctgact actcaattgc atttgtaaaa agtaaaacaa aattgtcaaa catatcactc tgtagtctqg acatataaca tagcttttat tatttaactg tc 2100 2160 2220 2280 2340 2400 2460 2512 <210> <211> <212> <213> <220> <221> <222> <223> 32 2512 DNA Arabidopsis thaliana promoter (2512) transcription regulating sequence from Arabidopsis thaliana gene At 4 gil490 <400> 32 tcgccttaaa atttcggaga tattctagtc acctatttag aaaagaatag tagtaagaac agagccgatc actaacaata agcattatct taagaaaaag tcgtttgatc tgtgaaaaca tttacaccct cagttgtagg ttatagaaga gggaatcaca tagcaaacac ttcctaccaa gtataaatgt aaacgacgag tgttaccttg gcttcttcaa t cagat tgt a attataagta atttgttgaa ctgcataact tcttttctat ataaaacata aatgtattta cttcacacca ctaggttgtg aacgtttaat gaacgtgaaa ggtaatttgc ccacgatcaa ttagcaatga cataatttct agggagcttt aaaaaaataa ttcaattgtt actctttcag tgtactaaac ccaaccgggg aaatttctta ttccattcca ttcttcgagt ccaggggcgg ttagcttgta acaatgtgaa cgtctttgga atatatatat gaagatttta cagcaaatga aaaaaaaact ggqcttaaaa attaataatc caaaacaaac agcaaaaaaa ggatgggtcg attgtcacta tgttttatgc ggtaaagggt ttgaggqcct tctccttgct tgcgacagcc cggttatata ttcacgattc tggaagttag atcagttctc gggaggatgc atctaggtgt aacttatgca attcaaaaat taatacatta atatatttat aagaatagaa aagtaacata ggtacgaacc catttttagt tataaaacat actatagaaa aagaaggaac ggcctgccaa gtccaaacag atccaactct tttgttgtaa attttgtaaa ccgttgagtt tcatgagctt atctgattat taccgttgtt ggtttcctgg gtagagcttc gtaatccatg atgggatgtg gtttaaatga aataataaaa ataccataca ttatttattt tgtatttacg cataattaca aaactgattt atgtggtctt gaaagggaaa cgtgaaaaga gaagtaagta tcaacaatag attacaactt atggtttagc tcttaaaact gaaaaatttt accgcactaa gaagttgttt gtataaccaa ccgcctaatt ccgttgattg taatgtcacg tgccaatagc gggcacgtgc ttcagcacaa aatcaaatgt ttttttcttt atttttaaaa acaaaaaaag tacaaaaagc accaatcaac tccaaaatat aaatactata aaaaaattgc gaagttgtgg aaaaatatat t tcaaagt tc atatgtggtc tggggcctat ctaaaacttt tgctcactac agcaaatcgg accggttatt ctgatttgtt gttcgttccc gctcgtgatg agagtcgtgc cctacattaa ctggttaagt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 PF 56134 gttttcaata tttttgactt tgatttatga gtatatgtgt gaaaatgaat aaattgtgag tttaataacg aatagtgccc ctgatggagt tcttgatgct taaggtcatt tggttttgta atattactaa tgattcgttg gtgaatatca catcagatca aatgccttgg atatgaagtg ttcgtccatc tgctcatttt acatcaatt attgatatat aaaaaagtta aaacaaatta aatagtatat cagattaaat tgtcttaatt tcaagtagcc ctttaatctt ttactgtttg t tt gggtat t tgttaagttg catcattaac ttgatttgat atattttaag tacggatatg cttacccagg ttcccaaat ttaactttct ttctaaaatg tatgaacaa cagaatttat ataataattt atttttctag agaaatatga tgtatttttt tactttaaac tgctgagaaa tggattgctc aggagattca atttttgaaa tggttatatg tttgacaaat tacatactaa ttcgagaata tttatcaatt tttaaaattt t t tga tat at atttcatagt tgaaatacat tgataaaaaa atcagccact aaatgqaaat tttaagtggg ccattgtaa gtaataagat aatttgttgg ttaaaagc attcaacatt att t tgtat a tgctgagaga cccaaaa gttgaagtaa taatatta atttattaat ctattcattt tt tt tt t tgg attttatcaa taatatataa ggattgtaac gtaatgatgt ggttcgaaac caatgcctat aagatattga ttatttaact atagtgggag cttttaataa tgctctgact actcaattga at t tgtaaa a cttttttatt tttattaatt tgattagata acttttatgt gtacctatat aaacttatag taagtaaatt atggaagcgt cagaaaatcg ttggattcag agtaaaaaa aattgtcaaa aatatatc tgtagtctgg aaatataaa t agct t ttat tatttaactg tc 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2512 <210> 33 <211> 1911 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (1)..(1911) <223> coding for serin/threonin kinase <400> 33 atg aga aag Met Arg Lys 1 ata agc ata Ile Ser Ile tat ttt gaa Tyr Phe Glu aca tat att Thr Ser Leu taa ata gga Ser Ile Gly 65 like protein ata tta tgg gtt Ile Phe Trp Val aaa aag aag att tac ttt Thr Lys Lys Ile Ser Phe 5 ttg Leu 10 caa Gin gtt ata Vai Leu att Ile aat Pro ggt gat att tat Giy Aia Ile Ser tag Ser 25 ga a Asp caa tga aaa Gin Cys Asn gaa aat ggg Giu Thr Giy tgg aaa aat Trp Lys Thr taa Tyr 40 gtg Val aaa aaa agt Thr Asn Arg agg aag ata att Arg Gin Ile Leu tta taa aat tat Phe Tyr Asn Ser gat taa aaa Ala Ser Lys gtg Val 55 gac Asp gat aa taa Asp His Tyr gga Gi y atg Met aaa gtt Lys Val aat Pro 70 gaa gtg aa Giu Val His gt a Val1 ggg atg tga Gly Met Cys I PF 56134 gao ggg acc gaa Asp Gly Thr Glu acg gtt tgc tcc Thr Val Cys Ser gat tgt Asp Cys caa act Gin Thr ctc aag gtc Leu Lys Val gcg gc Ala Ala gac caa tta Asp Gin Leu caa Gin 100 aag Lys gag aat tgt cct Glu Asn Cys Pro aca cct cat Thr Pro His 115 acg ctc tgt Thr Leu Cys ttt Phe 120 oca Pro aac Asn 105 gct Ala ctt Leu gaa gcg Glu Ala tat aca tgg Tyr Thr Trp 110 agt tca ttc Ser Ser Phe cgt tac tct Arg Tyr Ser tao atg gag Tyr Met Glu aac Asn 125 tto aag Phe Lys 130 gat ato Asp Ile 145 agg Arg gtt gga ttg Val Gly Leu cac His 135 aca Thr aaa tca aat ttg Lys Ser Asn Leu tat tta aat acg Tyr Leu Asn Thr 155 140 ata Ile cat agt aat gtg His Ser Asn Val tgg gag got ota Trp Giu Ala Leu 160 acg gat cgt ttg Thr Asp Arg Leu tot gao goa too Ser Asp Ala Ser tcg Ser 170 gat tat aat goa tca tta Asp Tyr Asn Ala Ser Leu 175 tct agt cgt Ser Ser Arg cag aat ata Gin Asn Ile 195 got tgt cac Ala Cys His 210 aga Arg 180 tat tat gca got Tyr Tyr Ala Ala gta aca aat otg Val Thr Asn Leu aca aat tto Thr Asn Phe
7. 190. gaa aaa ggt Glu Lys Gly tat goa tta atg Tyr Ala Leu Met cta Leu 200 tgc act cot gat Cys Thr Pro Asp ota Leu 205 576 624 672 aao tgt otg Asn Cys Leu gaa Glu 215 aaa got gtt tot Lys Ala Val Ser tat ggc aac ott Tyr Gly Asn Leu agg Arg 225 atg oaa aga gga Met Gin Arg Gly gtt gca tgg oca Val Ala Trp Pro ago Ser 235 tgo tgt ttt ogg Cys Cys Phe Arg tgg Trp 240 gat otg tat ccc Asp Leu Tyr Pro tto Phe 245 ato gga got ttt Ile Gly Ala Phe aat Asn 250 ttg aca ott toa Leu Thr Leu Ser ccc ccg Pro Pro 255 oca ggt ago Pro Gly Ser gtt gc aco Val Ala Thr 275 ott gtt tgo Leu Val Cys 290 aaa Lys 260 agg aat ato toa Arg Asn Ile Ser gga tto ttt gtg Gly Phe Phe Val gc att gtt Ala Ile Val 270 gta gta gta Val Val Val gga gtt gto ato Gly Val Val Ile tct Ser 280 gtg ota tot act Val Leu Ser Thr 816 864 912 aga aag aga Arg Lys Arg act gat cot oca Thr Asp Pro Pro gag Glu 300 gaa tca cot aaa Glu Ser Pro Lys tat Tyr 305 toa otg cag tat Ser Leu Gin Tyr gat Asp 310 ott aag aca att Leu Lys Thr Ile got gca aca tgt Ala Ala Thr Cys aco 960 Thr 320 r PF 56134 U ttt tea aag tgc Phe Ser Lys Cys atg ctt ggt caa Met Leu Gly Gin ggt gga Gly Gly 330 ttt gga gaa gtt ttc Phe Gly Giu Vai Phe 335 aag ggt gtg Lys Gly Val aaa gaa tca Lys Giu Ser 355 ctt Leu 340 gac gga tea Asp Gly Ser gaa Glu 345 att gca gtg aag Ile Ala Val Lys gct caa ggt gta Ala Gin Gly Val gag tte cag aat Glu Phe Gin Asn gag Glu 365 ctc Leu agg ctg tea Arg Leu Ser 350 act agt ctc Thr Ser Leu ggg ttt tgt Gly Phe Cys gtg gca Val Ala 370 aag ctt eag cac Lys Leu Gin His aga Arg 375 aat ttg gtt gga Asn Leu Vai Gly atg Met 385 age Ser gaa gga gaa gaa Glu Gly Giu Glu aag Lys 390 ttg Leu ata etc gta tac Ile Leu Val Tyr gaa Glu 395 aag Lys ttt gtt ccc aac Phe Val Pro Asn aaa Lys 400 etc gac eag Leu Asp Gin tte Phe 405 ttt gaa cct Phe Giu Pro aca Thr 410 aaa ggc caa Lys Gly Gin tgg geg aaa egg Trp Aia Lys Arg 420 tat ett eat eat Tyr Leu His His tac aag att att Tyr Lys Ile Ile gtt Val 425 aaa Lys gga act get aga Gly Thr Aia Arg gga Gly 430 gac Asp etg gat Leu Asp 415 att eta Ile Leu etc aaa Leu Lys gac tea ccc Asp Ser Pro get agt Ala Ser 450 ttt gga Phe Gly 435 aae Asn etc Leu 440 gct Ala ate ata eac Ile Ile His egt Arg 445 aaa Lys 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 ate etc tta Ile Leu Leu gat Asp 455 ttt Phe gaa atg gaa Glu Met Glu gte gea gat Val Ala Asp atg gea aga Met Ala Arg agg gtg gat Arg Val Asp 465 aga Arg caa Gin 475 tct Ser ega geg gat Arg Ala Asp agg gta gtt Arg Val Val gga Gly 485 tcg Ser eat gge tae His Gly Tyr eca gag tat Pro Giu Tyr ttg atg Leu Met 495 gte ttg Val Leu cat ggc cag His Gly Gin gtt ctt gag Val Leu Glu 515 gat gaa tee Asp Giu Ser gtg aaa tct Val Lys Ser gat Asp 505 tat agt ttt gga Tyr Ser Phe Gly ata agt gga Ile Ser Gly aaa Lys 520 gte Val aga aac age aac Arg Asn Ser Asn tte Phe 525 agg Arg 510 eat gaa act His Giu Thr eat tgg aga His Trp Arg gga aag aat Gly Lys Asn 530 aae gga Asn Gly 545 ttg Leu 535 ctt Leu aca tat get Thr Tyr Ala tgg Trp 540 tea eca tta Ser Pro Leu gag Glu 550 gtg gat tea Val Asp Ser gaa Glu 555 etc gaa aag aat Leu Glu Lys Ase PF 56134 cag agt aat Gin Ser Asn gaa gtc ttc aga tgc atc Glu Val 565 Phe Arg Cys Ile cat His 570 ate gcg cta tta tgt gtt Ile Ala Leu Leu Cys Val 575 atg atg Met Met caa aat gat Gin Asn Asp ctc aca agt Leu Thr Ser 595 gag gga atg Glu Gly Met 610 gtc aac gat Val Asn Asp 625 gaa caa cgt ccg Glu Gin Arg Pro aat Asn 585 tta tct act atc Leu Ser Thr Ile aac tcc atc act Asn Ser Ile Thr ccg gtg cct cag Pro Val Pro Gin ccg gta tat Pro Val Tyr cct ggt tct Pro Gly Ser 1728 1776 1824 1872 1911 gac atg ttt Asp Met Phe tca ttg att Ser Leu Ile 630 cta Leu 615 gat Asp tct atc aaa Ser Ile Lys tct ctt Ser Leu 620 cgc tga Arg gac tta gtt cct Asp Leu Val Pro 635 <210> 34 <211> 636 <212> PRT <213> Arabidopsis thaliana <400> 34 Met Arg Lys Thr Lys 1 5 Lys Ile Ser Phe Ile Phe Trp Val Val Leu Ile Ser Ile Tyr Phe Glu Ile Gly Ala Ile Ser Gin Gin Cys Asn Glu Thr Gly Gin Ile Leu Pro Trp Lys Thr Tyr Asp Thr Asn Arg Thr Ser Leu Ala Ser Lys Val 55 Val Asp His Tyr Gly Phe Tyr Asn Ser Ser Ile Gly Lys Val Gly Thr Glu Pro Asp Glu Val His Val Met Gly Met Cys Asp Thr Val Cys Ser Cys Leu Lys Val Ala Ala Asp Gin Leu Thr Pro His 115 Gin 100 Glu Asn Cys Pro Asn 105 Gin Thr Glu Ala Tyr Thr Trp 110 Ser Ser Phe Lys Thr Leu Cys Phe 120 Ala Arg Tyr Ser Asn 125 PF 56134 Phe Lys Arg Val Gly Leu His Pro Leu Tyr Met Glu 140 Ile His Ser Asn Val Trp Giu Ala Leu Thr Asp 145 Thr Lys Ser Asn Tyr Leu Asn Thr 155 Asp 160 Asp Arg Leu Met 165 Tyr Asp Ala Ser Ser 170 Val Tyr Asn Ala Ser Leu 175 Ser Ser Arg Gin Asn Ile 195 Ala Cys His 210 Arg 180 Tyr Tyr Ala Ala Thr Asn Leu Ala Leu Met Leu 200 Lys Thr Pro Asp Leu 205 Tyr Thr Asn Phe 190 Giu Lys Gly Gly Asn Leu Asn Cys Leu Glu 215 Val Ala Val Ser Arg 225 Asp Met Gin Arg Gly Ala Trp Pro Ser 235 Leu Cys Phe Arg Trp 240 Leu Tyr Pro Phe 245 Arg Gly Ala Phe Asn 250 Gly Thr Leu Ser Pro Pro 255 Pro Gly Ser Val Ala Thr 275 Leu Val Cys 290 Tyr Ser Leu Lys 260 Gly Asn Ile Ser Phe Phe Val Val Val Ile Ser 280 Thr Leu Ser Thr Leu 285 Glu Ala Ile Val 270 Val Val Val Ser Pro Lys Arg Lys Arg Gin Tyr Asp 310 Cys Asn Met Lys 295 Leu Asp Pro Pro Glu 300 Ala Lys Thr Ile 305 Phe Glu 315 Gly Ala Thr Cys Ser Lys Leu Gly Gin Phe Gly Glu 325 Gin Val Phe 335 Leu Ser Lys Gly Val Asp Gly Ser Glu 345 Ala Val Lys Arg 350 Lys Giu Ser Ala Gin Gly Val Gin Giu Phe Gin Asn Giu Thr Ser Leu 355 360 365 PF 56134 Val Ala 370 Lys Leu Gin His Arg 375 Asn Leu Val. Giy Leu Gly Phe Cys Met 385 Giu Gly Giu Giu Lys Ile Leu Val. Tyr Glu Phe Val. Pro Asn Ly's 400 Leu Asp 415 Ser Leu Asp Gin Phe Leu Phe Giu Pro Thr Lys Lys Gly Gin Trp Ala Lys Arg 420 Tyr Lys Ile Ile Gly Thr Aia Arg Giy Ile Leu 430 Asp Leu Lys Tyr Leu His His Asp Ser Pro Leu Lys Ile Ile His Arg 445 Aia Ser 450 Asn Ile Leu Leu Aia Giu Met Giu Lys Vai Ala Asp Phe 465 Gix' Met Ala Arg Phe Arg Vai Asp Gin 475 Ser Arg Ala Asp Arg Arg Vai Vai Gly 485 Thr His Gly Tyr Ser Pro Giu Tyr Leu Met 495 His Giy Gin Val Leu Giu 515 Ser Vai Lys Ser Asp 505 Vai Tyr Ser Phe Giy Val. Leu 510 His Giu Thr Ile Ile Ser Gly Lys 520 Arg Asn Ser Asn Asp Giu 530 Ser Gly Lys Asn Vai Thr Tyr Ala T rp 540 Arg His Trp Arg Asn 545 Gly Ser Pro Leu Ser Asn Giu Val 565 Giu 550 Leu Val. Asp Ser Leu Giu Lys Asn Gin Phe Arg Cys Ile His 570 Ile Ala Leu Leu Cys Val 575 Gin Asn Asp Pro 580 Giu Gin Arg Pro Asn 585 Leu Ser Thr Ile Ile Met Met 590 Pro Vai Tyr Leu Thr Ser Asn Ser Ile Thr 595 Leu 600 Pro Val. Pro Gin Ser 605 PF 56134 Glu Gly Met Asp Met Phe Leu Pro Ser Ile Lys Ser Leu Pro Gly Ser 610 615 620 Val Asn Asp Ser Leu Ile Asp Asp Leu Val Pro Arg 625 630 635 <210> <211> 1854 <212> DNA <213> Arabidopsis thaliana <220> <221> <222> <223> promoter (1854) transcription regulating sequence localized downstream and in opposite direction from Arabidopsis thaliana gene At2g31160 <400> tcgagtcatt tgttttatcc ctcaataaaa acaagacaag cattacacaa tcatttatgt gtttaaggta ttgtttcatc atataccaaa ttcaaatata aataaaatca atatgttttc ttaaactctc tttgcaaata cacaaataaa cgatttttga aaaaactggt gtaggattac atgccgtatt catgtttcag tccataaaat tgtagtattt aattttgaat attaatttta ccttcacaag gagttaaata ttcaattcat tgtcaaatga gttgaagggc tgagagacag cgtaccaaga acacataatg ctaaaaaaat cgccggacct tgagataaaa attttattta gataccccat tctttctctt actttagcag tatatcacaa tgaaaagaat aaaacatatt taagatatac cat t tgt tt t gctcttagaa aattgcggct aaattaatat tttttctttg actatgtata aaatataaga ggtgttatgt tctcqtcgct cctgaaactt attttacaat tggggtcttt aactctggct tgagtattat aggatatcaa gtgataacat attgttctga atctgtccaa aatattaaat tacaacaaaa tctctctata ccaaaactac aatatgatgt ggtgcaactt ctatgcatct taggtaaatt tttcattttt aagttgacat agcaatcatg tt tt tt t aca ttgttaataa t ct tgt act t tcattagaca catatatgtt ataattgtat taggaaaatt atgtggaagg gagtqctctg ctacctctaa tttaaaagag gatagcaaaa atgttgtttg cgctatctaa tcaaaacttt aaattcatta aaaaaccagc cacatagtta aaaattttat acgaacctta gcataatgga tcattagaat tcagttttct ttcgtaggat catgtaggca tggcaatctt tcgttgattt gaatgtggta gtgaaaaaat tcacataaaa aagttagtga ttaaatgaca gaaaggtaga aaattcagaa ggagtaaaca cacactcatg attttagtat gtatcacata attatcaaat actattatat ttcaagttgt gcttatgaaa aaaatatata tatacttata gagatctata tttaattata agaagaagat agcataccaa tatttataat taccattact actactagag tccgtattag gataactata aacaatggta ataagtaggt ttagtctttc aaaccgtagt tacttagtgt actacagaat atggacaaga acttacagag aatgatgcaa tagttatatt tat ggt ct aa ttgacaaaga tgattaaaac atttgtcqtt attaccccca ctgaaaaatg tatatacagt attttcacaa taattgacaa catagacatt aacttattta tccgctattc aatcaaaatg attccatctt gcaacttaac aacgatgagt tactaatgcc catttcacca ctcttgttgg tactacaaat cgctataatt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 PF 56134 gtattgaaat gacatactta gtggcacaca atctatttaa catggaatta ccataattaa aatacaacat caatcatgaa tcatcagatc tacaaaatct agggtttatg caaaag ccga gacacgcaca ttgttcttct tgagtaaaaa agtaagtact aagaaacata aagtggagaa tttacggaac tgtttttttt tccctacaat tatgctgtaa gagaacatat cactcacaac tcacaagtaa acctatgaaa cctactttaa catgaaaaag aaatgatttt agag <210> 36 <211> 1855 <212> DNA <213> Arabidopsis thaliana 1620 1680 1740 1800 1854 <220> <221> <222> <223> promoter (1855) transcription regulating sequence localized downstream and in opposite direction from Arabidopsis thaliana gene At2g31160 <400> 36 tcgagtcatt tgttttatcc ctcaataaaa acaagacaag cattacacaa tcatttatgt gtttaaggta ttgtttcatc atataccaaa ttcaaatata aataaaatca atatgttttc ttaaactctc tttgcaaata cacaaataaa cgatttttga aaaaactggt gtaggattac atgccgcatt catgtttcag tccataaaat tgtagtattt aattttgaat attaatttta ccttcacaag gagttaaata gtattgaaat ttcaattcat tgtcaaatga gttgaagggc tgagagacag cgtaccaaga acacataatg ctaaaaaaat cgccggacct tgagataaaa attttattta gataccccat tctttctctt actttagcag tatatcacaa tgaaaagaat aaaacatatt taagatatac catttgtttt gctcttagaa aattgcagct aaattaatat tttttctttg actatgtata aaatataaga ggt gt tat gt tctcgtcgct gacatactta cctgaaactt attttacaat tggggtcttt aactctggct tgagtattat aggatatcaa gtgataacat attgttctga atctgtccaa aatattaaat tacaacaaaa tctctctata ccaaaactac aatatgatgt ggtgcaactt ctatgcatct taggtaaatt tttcattttt aagttgacat agcaatcatg tttttttaca ttgttaataa tcttgtactt tcattagaca catatatgtt ataattgtat gtggcacaca taggaaaatt atgtggaagg gagtgctctg ctacctctaa tttaaaagag gatagcaaaa atgttgtttg cgctatctaa tcaaaacttt aaattcatta aaaaaccagc catatagtta aaaattttat acgaacctta gcataatgga tcattagaat tcagttttct ttcgtaggat catataggca tggcaatctt tcgttgattt gaatgtggta gtgaaaaaat tcacataaaa aagttagtga ttaaatgaca atctatttaa gaaaggtaga aaattcagaa ggagtaaaca cacactcatg attttagtat gtatcacata attatcaaat actattatat ttcaagttgt gcttatgaaa aaaatatata tatacttata gagat ct ata tttaattata agaagaagat agcataccaa tatttataat taccattact actactagag tccgtattag gataactata aacaatggta ataagtaggt ttagtctttc aaaccgtagt tacttagtgt catggaatta actacagaat atggacaaga acttacagag aatgatgcaa tagttatatt tatggtctaa ttgacaaaga tgattaaaac atttgtcgtt attaccccca ctgaaaaatg tatatacagt attttcacaa taattgacaa catagacatt aacttattta tccgctattc aatcaaaatg attccatctt gcaacttaac aacgatgagt tactaatgcc catttcacca ct ct tgt tgg tactacaaat cgctataatt ccataattaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 PF 56134 aatacaacat caatcatgaa tcatcagatc tacaaaatct agggtttatg caaaagccga gacacgcaca ttgttcttct tgagtaaaaa agtaagtact aagaaacata aagtggagaa tttacggaac tgtttttttt tccctacaat tatgctgtaa gagaacatat cactcacaac tcacaagtaa acctatgaaa cctactttaa catgaaaaag aaatgatttt agagc 1680 1740 1800 1855 <210> -C211> <212> <213> 37 DNA Artificial <220> <223> Oligonucleotide primer <400> 37 ggcgctcgag tattgaaata aaatcagttg <210> 38 <211> 34 <212> DNA <213> Artificial <220> <223> Oligonucleotide primer <400> 38 cggccatgga aggtgtatat atagagatta cttc <210> 39 <211> 32 <212> DNA <213> Artificial <220> <223> Oligonucleotide primer <400> 39 gcggatccgc ctccatagga tgctcatgct gt 32 <210> <211> <212> <213> <220> <223> 27 DNA Artificial Oligonucleotide primer PF 56134 <400> gagccatggc tacacgagtc agattcc 27 <210> 41 <211> 39 <212> DNA <213> Artificial <220> <223> Oligonucleotide primer cN~1<400> 41 cgcggatcca tttcctttaa agagaataat ttaagttaa 39 <210> 42 <211> <212> DNA <213> Artificial <220> <223> Oligonucleotide primer <400> 42 gcgccatggc gtttaggttt tgtgtttaaa attcg <210> 43 <211> 33 <212> DNA <213> Artificial <220> <223> Oligonucleotide primer <400> 43 cgccatggag gaaaaatgga agaggaagag ttc 33 <210> 44 <211> 36 <212> DNA <213> Artificial <220> <223> Oligonucleotide primer PF 56134 56 <400> 44 cgcgqatcct gttttgggag atgcattcaa taaaga <210> <211> 36 <212> DNA <2,43> Artificial <220> <223> Oligonucleotide primer <400> gcgccatggt ctcaaaccaa caaatcttgt agccac <210> <211> <212> <213> 46 29 DNA Artificial <220> <223> Oligonucleotide primer <400> 46 cgcggatcca taatcaacct tctcgtttg <210> 47 <211> <212> DNA <213>- Artificial <220> <223> Oligonucleotide primer <400> 47 gcgccatggc agaaagtgaa gatctcgact aagag <210> <211> <212> <213> <220> <223> 48 27 DNA Artif icial Oligonucleotide primer <400> 48 PF 56134 57 I cgctcgagta atcaaccttc tcgtttg 27 CK <210> 49 U <211> <212> DNA <213> Artificial 0\ <220> <223> Oligonucleotide primer a\ <400> 49 cgccatggcc tccctctctg cgcctcctgg <210> 0 15 <211> 29 <212> DNA <213> Artificial <220> <223> Oligonucleotide primer <400> cgcggatcca ttataactct atgttgcat 29 <210> 51 <211> <212> DNA <213> Artificial <220> <223> Oligonucleotide primer <400> 51 gcgccatgga gtctagatat atattgcgag ttgtc <210> 52 <211> 29 <212> DNA <213> Artificial <220> <223> Oligonucleotide primer <400> 52 cgcggatccc ttcttcttac gtttccagt 29 PF 56134 <210> <211> <212> <213> 53 DNA Artificial <220> <223> Oligonucleotide prinmer <400> 53 gcgccatggc agtaagatta ttgtaagaga aatgc <210> 54 <211> 37 <212> DNA <213> Artificial <220> <223> Oligonucleotide primter <400> 54 cgcggatcct tttgacgaat atacaaatac gtttctt <210> <211> 42 <212> DNA <213> Artificial <220> <223> Oligonucleotide primer <400> gcgactagtt gcaaataaca tttaaaaaag aaagaaaaag ag 37 <210> 56 <211> 29 <212> DNA <213> Artificial <220> <223> Oligonucleotide primer <400> 56. gcgctcgagt tcgccttaaa atttgttga PF 56134 59 in <210> 57 <211> C1 <212> DNA <213> Artificial <220> <223> Oligonucleotide primer <400> 57 0 10 cgcggatccg attttacaaa tgtgtttggg gttag <210> 58 C1 <211> <212> DNA S 15 <213> Artificial <220> <223> Oligonucleotide primer <400> 58 agagataaat actcgagtca ttttc <210> 59 <211> 27 <212> DNA <213> Artificial <220> <223> Oligonucleotide primer <400> 59 ccgccatggc tctaaaatca tttcttt 27 <210> <211> 8986 <212> DNA <213> Artificial <220> <223> binary vector pSUN0301 <400> cgttgtaaaa cgacggccag tgaattcgag ctcggtacct cgagcccggg cgatatcgga tccactagtc tagagtcgat cgaccatggt acgtcctgta gaaaccccaa cccgtgaaat 120 caaaaaactc gacggcctgt gggcattcag tctggatcgc gaaaactgtg gaattggtca 180 PF 56134 gcgttggtgg gaaagcgcgt cgatcagttc gccgatgcag agtctttata ccgaaaggtt tcattacggc aaagtgtgqq gccatttgaa gccgatgtca gcttctacct ttgatatata aatatttttt tcaaaataaa gtgtgtatat tttaatttat tatcaccgtt tgtgtgaaca taccgacgaa aacggcaaga aatccatcgc agcgtaatgc ggtgacgcat gtcgcgcaag tggtgatgtc agcgttgaac cactagcggg actttgcaag ctatgaactg tgcgtcacag cggcatccgg tcagtggcag ctttactggc tttggtcgtc gctgatggtg cacgaccacg gcattaccct tacgctgaag tgatgaaact gctgctgtcg caagccgaaa gaactgtaca acaggcgatt aaagagctga tattgccaac gaaccggata agcaacgcgt aaactcgacc cgctcacacc gataccatca atggtatgtc caaagcggcg ggcctggcag qagaaactgc agccggqctg cactcaatgt ggatatgtat caccgcgtct tttcgccgat tttgcgacct cttcactcgc gaccgcaaac catgaacttc ggtgaaaaac cgcaccatcg tcggctacag tcaccagtct ctctctacaa agttcccaga *taagggaatt gaaaccctta gtatgtattt taaaaccaaa atccagtgac ttggcgtaat catggtcata gtttaaacta tcagtgtttg ttattagaat aatcqgatat atgtccatga taagtcgcgc ttttcatggc ttgttatgac agcaagcgcg ttacgccgtg agcagggcag tcgccctaaa tatcgactca actatcagag tacaagaaag atattcgtaa gggcaggcca tcaataatca cgccgtatgt tataataatt agaatgtagt aacttttcta acgaactgaa aaaagcagtc tctacaccac actgtaacca tgcgtgatgc tggtgaatcc ccaaaagcca tgaagggcga atgaagatgc cattaatgga agatgctcga gctttaacct gcgaagaggc tagcgcgtga cccgt ccgca cgacgcgtcc gcgatctctt atttggaaac atcagccgat acaccgacat ttgatcgcgt cgcaaggcat cgaagt agga cgcagcaggg cctcgggaat atctatctct agggttctta gtatttgtaa cgggtaccga gctgtttcct acaggatata ttaaaagggc tgtatgtgtt t gt ttt t ttg ggtcgatgtt acaaagttaa gtagttggcg *ccgggcaatt ttatgcgggc gcgtatcgtg ggaagtgatg tattgccggg atcattaatt atatagcaat atatatgacc ctggcagact ttacttccat gccgaacacc cgcgtctgtt ggatcaacag gcacctctgg gacagagtgt acagttcctg ggacttacgt ctggattggg ctgggcagat ctctttaggc agtcaacggg caaaaaccac agtgcacggg gatcacctgc tgatgtgctg ggcagagaag tatcatcacc gtggagtgaa cagcgccgtc attgcgcgtt ggcttttctg aggcaaacaa tgctaccgag ctctattttt tagggtttcg aatacttcta gctcgaattt actagatctg ttggcgggta gtgaaaaggt tgtttgaata gggtacagtc tgatgttatg acatcatggg tcatcgagcg *gctgtgccag aacgtctggt Ictgcgtttcg *gagcatcagg *aaaagtgtac agtagtaata t gct tt tct g aaaatttgtt atcccgccgg gatttcttta tgggtggacg gactggcagg gt ggt tgca a caaccgggtg gatatctacc attaaccaca ggcaaaggat gccaactcct gaacatggca attggtttcg gaaactcagc ccaagcgtgg aatatttcgc gtcaatgtaa tgcctgaacc gtactggaaa gaatacggcg gagtatcagt gtcggtgaac ggcggtaaca ctgcaaaaac tgaatcaaca ctcggtaccc ctccagaata ctcatgtgtt tcaataaaat cgacctgcag attgtcgttt aacctaagag t tat ccgt tc ttcatggaac tatgcctcgg gagcagcaac ggaagcggtg ccatctcgaa gcagttttaa atcagcgcga atgcggtcac gcggctatac gtaagtttct taatatttca tagtttataa gatgtgcagg gaatggtgat actatgccgg atatcaccgt tggtggccaa ctggacaagg aaggttatct cgcttcgcgt aaccgttcta tcgataacgt accgtacctc tcgtggtgat aagcgggcaa aagcgcactt tgatgtggag cactggcgga tgttctgcga gttattacgg aagaacttct tggatacgtt gtgcatggct aggtatggaa agaaagggat gctggactgg actctcctgg ggcgcaaaaa atgtgtgagt gagcatataa ttctaattcc gcatgcaagc cccgccttca aaaagagcgt gtccatttgt gcagtggcgg gcatccaagc gatgttacgc atcgccgaag ccgacgttgc 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 PF 56134 tggccgtaca atttgctggt accttttgga ccattgttgt ttqgagaatg ttgatctggc cggoggagga ccttaacgct cgttgtcccg ccgactgggc aggcttatct ttgtccacta attcgttcaa cacataattg cataataagc agttttcgtt ctttttttct tttgtttgcc cgcagatacc ctgtagcacc gcgataagtc ggt cgggctg aactgagata cggacaggta ggggaaacgc gatttttgtg tt t ta cggtt ctgattctgt gaacgaccga ttctccttac gcggcggggc gtgcgctggc aaagagtttt gaccggttcc ggctttgggt cctgctaggg ccctcgatca ttcaaatcgt ttcttgaact tctgccttgc atcaaaaagt cggtacatcc acgatcttgt ttggccttct accaggtcgt *tttgtacggc tacggtgacc *aacttcggct gcacgacgac *gcagcgcaat tatcttgctg actctttgat atggaactcg catttggtac aatggagcgc tggacaagaa cgtgaaaggc gccgacgccg ctcacagcca cctacacaaa ccactgagcg gcgcgtaatc ggatcaagag aaatactgtc gcctacatac gtgtcttacc aacggggggt cctacagcgt tccggtaagc ctggtatctt atgctcgtca cctggcdttt ggataaccgt gcgcagcgag gcatctgtgc gtagggagcg cagacagtta aggcggaaaa caatgtacgg tcccaatgta caatttgccc ggttgcggta actccggcag ctccggcgct ctgcggcgcg aatcggggtg aatcagctag agcggctaat tcgtacgctg ctttctgctt tccgcagtgg gtaaggcttg tcccctggag atcattccgt gacattcttg acaaaagcaa ccggttcctg ccgcccgact agcgcagtaa ctgccggccc gaagatcgct gaqatcacca cttcgcggcg aactatcagg ttgggagata tcagaccccg tgctgcttgc ctaccaactc cttctagtgt ctcgctctgc gggttggact tcgtgcacac gagctatgag ggcagggt cg tatagtcctg ggggggcgga tgctggcctt attaccgcct tcagtgagcg ggtatttcac cagcgaccga tgcacaggcc atcgcctttt ctttgggttc cgtgctatcc tagcatctgc gcgcatgact gtcatttgac gccactgcgt gcgtgccagg aaccgtcagc ctcgatctcg caaggcttca catggcaacg tccgccatcg atggcggcct atgaaacaac agagcgagat ggcgttatcc caggtatctt gagaacatag aacaggatct gggctggcga ccggcaaaat agtatcagcc tggcctcgcg aggtagtcgg cggcttaact tcaagtctgc tatcatgcat tagaaaagat aaacaaaaaa tttttccgaa agccgtagtt taatcctgtt caagacgata agcccagctt aaagcgccac gaacaggaga tcgggtttcg gcctatggaa ttgctcacat ttgagtgagc aggaagcgga accgcatagg agggtaggcg aggcgggttt ttctctttta ccaatgtacg acaggaaaga tccgtacatt aggatcgggc ccgatcagct tcgtagatcg cggtagagaa acgtccgggt atgtactccg ccctcggata tgcgtggtgt gctcgccggc gaagccacac gcggcgagct tctccgcgct agctaagcgc cgagccagcc cgttgccttg atttgaggcg tgagcgaaat cgcgccgaag cgtcatactt cgcagatcag caaataatgt caagcgttag ttttattatt gaccaaaatc caaaggatct accaccgcta ggtaactggc aggccaccac accagtggct gttaccggat ggagcgaacg gcttcccgaa gcgcacgagg ccacctctga aaacgccagc gttctttcct tgataccgct agagcgcctg ccgcgatagg ctttttgcag taagagtttt tatcagtcac ggttccggtt gaccttttcg aggaaccggc cagcctgccc tgcgcacggt tcttgaacaa aacggccgat tcttgccttc gccgcccggt ccgtcaccag ttaaccgaat agaacttgag agtgatattg ttgatcaacg gtaqaagtca gaactgcaat acgatcgaca gtaggtccag ctaaatgaaa gtagtgctta gatgtcgctg gaagctagac ttggaagaat ctagctagaa atgcactaag tttaagcgtg ccttaacgtg tcttgagatc ccagcggtgg ttcagcagag ttcaagaact gctgccagtg aaggcgcagc acctacaccg gggagaaagg gagcttccag cttgagcgtc aacgcggcct gcgttatccc cgccgcagcc atgcggtatt ccgacgcgaa ctcttcggct aataagtttt ttacatgtgt cccaatgtac acctttttcc ggatgcttcg cgcctcctcc gaaacagaac ccatctggct gccgggatcg tgtgatctcg ttcgctcttt gcggccgttc gcaggtttct tacgtccgca 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 PF 56134 acgtgtggac gattcggtta coggoaggc ccagctcgtc tcgcgggtgc ggcggcttcc cgatcagcgg tgggcggcct tgtaccgggc tgccattgca cacacatggg ctttagccgc gcgatgtatt agcttggtgt gccaggctgg gtgtttgtgc tttcagcggc cggttgtgcc gaatgggcag tgatcgcccg gcttaaccag gaatcagcac ctccgtcgat ggcggtcgat cactgccctg gggctagatg taaccttcat gaccgcatqa t tt gt gccga gtaaacaaat attaacatcc aacaatctaa gaaggcggga ccgccgatga ggagccactc ttattgcgcg aatgctccac ctcaatccaa gcacgcaggt gacaatcggc ttttgtcaag atcgtggctg qggaagggac tgctcctgcc ggaacacgcg gatgggaaac ctgcggaaac gqtcacgctt ccacgtcata taatcgacgg ccccttgcca gcgcggcctt cggatggttt gggccggcag gcattccacg taaaattcat cagatagcag gatcctccgc ccaacgttgc ttttgctcat cagcgcctgg ggcggcggca ctcgtacccg cgacacgaca ctccaccagg gaagtcggct cactacgaag gccgacaacg gggatcggaa ggttgcgatg gcgttcccct cgcaagctgt gctgccggtc tgacgcttag gtttgatact tgacaattat aacgacaatc cgcgggacaa agccgcgggt ttcaaaagtc tgacgttcca ataatctgca tctccggccg tgctctgatg accgacctgt qccacgacgg tggctgctat gagaaagtat gccgggcttg cgccatcagt ctctacgtgc cgacagacgg gagcatcgga cgcaccggct cgattcaccg caacttctcc gcgaccgctc acaacccagc gcgtcggtgc ctactcattt ctcggtaatg cggcaactga agccttgctg tttctcttta acctcgcgqg gtgcctgggt gccagcgcct aaggccgctt tcggcggtgg gccttgatcg tcgcgccggc gttagcggtt tcgactaaca gtcgtcttgc tgcgtatttg tttactcaaa ggggagctgt acaacttaat tgtctaaaat taccaagcag tgatcatgag gccgttttac ttctggagtt gcctaaggtc taaattcccc ccggatctgg cttgggtgga ccgccgtgtt ccggtgccct gcgttccttg tgggcgaagt ccatcatggc tctcccttcc accaggtcgt ccgtctggaa aaaacggcca acgaaaaaat gccggcggtt gggcgtgctt accaggtcat acgccgattc cgcttacgcc ctggttgttc attcatttgc gtcttgcctt aagttgaccc ctgcgtgcgc cctcattaac cagcgtcgcc agct cacgcg cggcaacctc gtagccttcc cccatatgtc cggacaca.gc cgatggcctt gatcttcccg gaacatcggc ctgacccgcc tttatttact tacacatcac tggctggctg aacacattgc tggctgattt tgatcctgtc cggagaatta gtttggaact taatgagcta actatcagct tcggtatcca atcgtttcgc gaggctattc ccggctgtca gaatgaactg cgcagctgtg gccgqqgcag tgatgcaatg cttcccqgta aatcccacac gctcgtagcg cgtccatqat ctggttgctc gccgggattc ctgcctcgat cacccagcgc ctcgggcttg tggccaaccg ttgattttcc tcatttactc ggcgtaccgc gcttcatggc tcggacggcc tcaaatgagt ctcgggttct ctgcgtgata accgccgatg atccgtgacc gtaagggctt caagtccgcc cacgtcgcgg cacggccgcc cccggcgagt t t tt ggt ta catcgcatca ctttttagac gtggcaggat gga cgt at tt cgagtgcatc aaacactgat agggagtcac gacagaaccg agcacatacg agcaaatatt attagagtct atgattgaac ggctatgact gcgcaggqgc caggacgagg ctcgacgttg gatctcctgt cggcggctgc tcggttcatg actggccatg gatcacctcg gctgcgacta gtcgcccttg tttgcggatt gcgttgccgc cgcgccgatt ggggttccag cccgttcctc atgccgcctc tggtagctgc gtacatcttc tggcgtgtct ggcacttagc tttgatttaa gattcaagaa cgggactcaa tcaatgcgct ggctgcaccg gcctggggcg tcaatcgtcg caatcgcggg tgcagggcgc agtacagcga tatacgcagc gcgtggtgat atattgtggt aatgtactga tatgcataaa agtttaaact gttatgaccc caacgttgaa tcagaaacca tcttqtcaaa catattcact aagatggatt gggcacaaca gcccggttct cagcgcggct tcactgaagc catctcacct atacgcttga 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500
8. 7560. 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg PF 56134 gatggaagcc agccgaactg acatggcgat cgactgtggc tattqctgaa cgctcccgat acccaagctc gatgatcccc cggtcttgcg catgtaatgc catttaatac ggtgtcatct ggtcttgtcg ttcgccaggc gcctgcttgc cggctgggtg gagcttggcg tcgcagcgca tagatcttgc gatcgttcaa atgattatca atgacqttat gcgatagaaa atgttactag atcaggatga t caaggcgcg cgaatatcat tggcggaccg gcgaatgggc tcgccttcta tgcgttcgga acatttggca tataatttct ttatgagatg acaaaatata atcgggcctc tctggacgaa catgcccgac ggtggaaaat ctatcaggac tgaccgcttc tcgccttctt tattttcgtg ataaagtttc gttgaattac ggtttttatg gcgcgcaaac ctgtcaagct gagcatcagg ggcgaggatc ggccgctttt atagcgttgg ctcgtgcttt gacgagttct gagttcccgc ttaagattga gttaagcatg attagagtcc taggataaat ctgagt ggctcgcgcc tcgtcgtgac ctggattcat ctacccgtga acggtatcgc tctgagcggg cacagacccg atcctgttgc taataattaa cgcaattata tatcgcgcgc 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 8986